rAAV-based compositions and methods

ABSTRACT

The invention relates to isolated nucleic acids and rAAV-based compositions, methods and kits useful for treating genetic diseases (e.g., alpha-1 antitrypsin deficiency).

RELATED APPLICATION

This application is a continuation under 35 U.S. C. § 120 of U.S.application Ser. No. 14/952,217, entitled “RAAV-BASED COMPOSITIONS ANDMETHODS FOR TREATING ALPHA-1 ANTI-TRYPSIN DEFICIENCIES”, filed Nov. 25,2015, which is a continuation under 35 U.S. C. § 120 of U.S. applicationSer. No. 14/113,118, entitled “RAAV-BASED COMPOSITIONS AND METHODS FORTREATING ALPHA-1 ANTI-TRYPSIN DEFICIENCIES” filed Oct. 21, 2013, whichis a National Stage filing under 35 U.S.C. § 371 of internationalapplication PCT/US2012/034446, filed on Apr. 20, 2012, and entitled“RAAV-BASED COMPOSITIONS AND METHODS FOR TREATING ALPHA-1 ANTI-TRYPSINDEFICIENCIES,” which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 61/477,671, entitled “RAAV-BASEDCOMPOSITIONS AND METHODS FOR TREATING ALPHA-1 ANTI-TRYPSIN DEFICIENCIES”filed on Apr. 21, 2011, the entire content of each application which isincorporated by reference herein.

FEDERALLY SPONSORED RESEARCH

This invention was made with government support under HL069877 andDK032520 awarded by National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods and compositions for treating geneticdisease using rAAV-based vectors.

BACKGROUND OF THE INVENTION

Numerous diseases are associated with inherited or somatic mutations. Inmany cases, these mutations are present in the transcript region ofgenes, the products of which control important physiological functionsincluding, for example, gene expression, cell signaling, tissuestructure, and the metabolism and catabolism of various biomolecules.Mutations in these genes, which are often only single nucleotide changes(e.g., non-sense mutations, missense mutations), can have negativeeffects on the expression, stability and/or function of the gene productresulting in alterations in one or more physiological functions.

A number of different mutations have been identified in the Alpha-1antitrypsin (AAT) gene. AAT is one of the primary circulating serumanti-proteases in humans. AAT inhibits a variety of serine proteinases,with neutrophil elastase being one of the most physiologicallyimportant, as well as inhibiting a number of metalloproteinases andother pro-inflammatory and pro-apoptotic molecules. AAT is normallyproduced within hepatocytes and macrophages, where hepatocyte-derivedAAT forms the bulk of the physiologic reserve of AAT.

Approximately 4% of the North American and Northern European populationspossess at least one copy of a mutant allele, known as PI*Z (Z-AAT)which results from a single amino acid substitution of lysine forglutamate at position 342 in the mature protein (position 366 in theprecursor protein). In the homozygous state, this mutation leads tosevere deficiency of AAT, and can result in two distinct pathologicstates: a lung disease which is primarily due to the loss ofantiprotease function, and a liver disease (present to a significantdegree in approximately 10-15% of patients) due to a toxic gain offunction of the Z-AAT mutant protein.

Investigational clinical gene therapy products for gene augmentation ofAAT have been developed as potential treatments for lung disease usingthe recombinant adeno-associated viral (rAAV) vectors. Researchers havealso applied genetic technologies in an effort to down-regulate thelevels of AAT mRNA. One approach was to utilize hammerhead ribozymesdesigned to cleave AAT mRNA at a specific site. Another approachinvolves the use of RNA interference to decrease levels of the mutantmRNA transcript.

SUMMARY OF THE INVENTION

Aspects of the invention relate to improved gene therapy-based methodsfor treating genetic disease. Some aspects of the invention relate toimproved gene therapy compositions and related methodology for treatinglung disease and/or liver disease using the recombinant adeno-associatedviral vectors. In some embodiments, the methods utilize rAAV (e.g.,rAAV9, rAAV2, rAAV1) based vectors for augmenting AAT expression. Insome embodiments, compositions and methods are provided for decreasingthe expression of Pi*Z mutant AAT protein. In such embodiments, thecompositions and methods are useful for halting and/or amelioratinghepatocellular damage and other tissue damage associated with the mutantAAT.

According to some aspects of the invention, the compositions and methodsare useful for knocking down PiZ protein while at the same timeincreasing levels of the M-AAT protein (the wild-type AAT protein). Insome embodiments, a non-toxic dual function vector is provided that iscapable of knocking-down Z-AAT while augmenting M-AAT. According to someembodiments, methods and compositions for long-term expression oftherapeutic miRNAs are provided that utilize the recombinantadeno-associated virus (rAAV) platform. In some embodiments, therapeuticcompositions and methods described herein take advantage of the miRNApathway by altering the seed sequence of natural miRNAs to target theendogenous AAT gene. In some embodiments, the methods are safer and lesstoxic than shRNA-based approaches.

According to other aspects of the invention, rAAV-based compositions andmethods are provided that simultaneously direct silencing agents to theliver to decrease Z-AAT expression and direct gene augmentation to othersites. However, in some embodiments, the liver is an optimal targettissue for augmentation. In some embodiments, a miRNA-based approach isprovided to stably down-regulate Z-AAT within hepatocytes. In someembodiments, the approach allows for simultaneous M-AAT geneaugmentation from the same rAAV gene delivery vector without seriousperturbation of the overall hepatic miRNA profile. In some embodiments,the specific vector used is a systemically delivered rAAV9-capsidderived vector. According to some aspects of the invention, thisapproach has broad utility in genetic disorders stemming from dominantnegative and gain of function mutations as well as for deliveringartificial miRNAs to be delivered in conjunction with therapeutic genes.

According to some aspects of the invention, isolated nucleic acids areprovided. In some embodiments, the isolated nucleic acids comprise (a) afirst region that encodes one or more first miRNAs comprising a nucleicacid having sufficient sequence complementary with an endogenous mRNA ofa subject to hybridize with and inhibit expression of the endogenousmRNA, wherein the endogenous mRNA encodes a first protein; and (b) asecond region encoding an exogenous mRNA that encodes a second protein,wherein the second protein has an amino acid sequence that is at least85% identical to the first protein, wherein the one or more first miRNAsdo not comprise a nucleic acid having sufficient sequence complementaryto hybridize with and inhibit expression of the exogenous mRNA, andwherein the first region is positioned within an untranslated portion ofthe second region. In some embodiments, the untranslated portion is anintron. In some embodiments, the first region is between the first codonof the exogenous mRNA and 1000 nucleotides upstream of the first codon.

In some embodiments, the isolated nucleic acids comprise (a) a firstregion encoding one or more first miRNAs comprising a nucleic acidhaving sufficient sequence complementary with an endogenous mRNA of asubject to hybridize with and inhibit expression of the endogenous mRNA,wherein the endogenous mRNA encodes a first protein; and (b) a secondregion encoding an exogenous mRNA that encodes a second protein, whereinthe second protein has an amino acid sequence that is at least 85%identical to the first protein, wherein the one or more first miRNAs donot comprise a nucleic acid having sufficient sequence complementary tohybridize with and inhibit expression of the exogenous mRNA, and whereinthe first region is positioned downstream of a portion of the secondregion encoding the poly-A tail of the exogenous mRNA.

In some embodiments, the isolated nucleic acids further comprise a thirdregion encoding a one or more second miRNAs comprising a nucleic acidhaving sufficient sequence complementary to hybridize with and inhibitexpression of the endogenous mRNA, wherein the third region ispositioned within an untranslated portion of the second region. In someembodiments, the untranslated portion is an intron. In some embodiments,the first region is between the last codon of the exogenous mRNA and aposition 1000 nucleotides downstream of the last codon. In someembodiments, the third region is between the first codon of theexogenous mRNA and a position 1000 nucleotides upstream of the firstcodon.

In some embodiments of the isolated nucleic acids, the first regionencodes two first miRNAs. In some embodiments, the first region encodesthree first miRNAs. In some embodiments, the third region encodes twosecond miRNAs. In some embodiments, the third region encodes threesecond miRNAs. In some embodiments, one or more of the first miRNAs havethe same nucleic acid sequence as one or more of the second miRNAs. Insome embodiments, each of the first miRNAs has the same nucleic acidsequence as one of the second miRNAs. In some embodiments, the secondprotein has an amino acid sequence that is at least 90% identical to thefirst protein. In some embodiments, the second protein has an amino acidsequence that is at least 95% identical to the first protein. In someembodiments, the second protein has an amino acid sequence that is atleast 98% identical to the first protein. In some embodiments, thesecond protein has an amino acid sequence that is at least 99% identicalto the first protein. In some embodiments, the second protein has anamino acid sequence that is 100% identical to the first protein.

In some embodiments of the isolated nucleic acids, the first protein isAlpha 1-Antitrypsin (AAT) protein. In some embodiments, the AAT proteinis a human AAT protein. In some embodiments, the AAT protein hassequence as set forth in SEQ ID NO: 1 or 2 or one or more mutationsthereof as identified in Table 1, e.g. SEQ ID NO: 3 or 4. In someembodiments, the first mRNA comprises a nucleic acid encoded by asequence as set forth in SEQ ID NOS: 5-16. In some embodiments, the oneor more miRNAs have a nucleic acid sequence encoded by a sequence fromthe group consisting of SEQ ID NOS: 17-19 and 21-23. In some embodimentsof the isolated nucleic acids, the exogenous mRNA has one or more silentmutations compared with the endogenous mRNA. In some embodiments, theexogenous mRNA has a nucleic acid sequence encoded by a sequence as setforth in SEQ ID NO: 20.

In some embodiments, the isolated nucleic acids further comprise aninverted terminal repeats (ITR) of an AAV serotypes selected from thegroup consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9,AAV10, AAV11 and variants thereof. In some embodiments, the isolatednucleic acids further comprise a promoter operably linked with theregion(s) encoding the one or more first miRNAs, the exogenous mRNA,and/or the one or more second miRNAs. In certain embodiments, thepromoter is a tissue-specific promoter. In certain embodiments, thepromoter is a β-actin promoter.

According to some aspects of the invention, recombinant Adeno-AssociatedViruses (AAVs) are provided that comprise any of the isolated nucleicacids disclosed herein. In some embodiments, the recombinant AAVsfurther comprise one or more capsid proteins of one or more AAVserotypes selected from the group consisting of: AAV1, AAV2, AAV3, AAV4,AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11 and variants thereof.

According to some aspects of the invention, compositions are providedthat comprise any of the isolated nucleic acids disclosed herein.According to some aspects of the invention, compositions are providedthat comprise any of the recombinant AAVs disclosed herein. In someembodiments, the compositions further comprise a pharmaceuticallyacceptable carrier.

According to some aspects of the invention, kits are provided thatcomprise one or more containers housing a composition, isolated nucleicacid or rAAV of the invention. In some embodiments, the kits furthercomprise written instructions for administering an rAAV to a subject.

According to some aspects of the invention, methods are provided forexpressing Alpha 1-Antitrypsin (AAT) protein in a subject. In someembodiments, the methods comprise administering to a subject aneffective amount of any recombinant Adeno-Associated Virus (rAAV)disclosed herein. In some embodiments, the rAAV is administered with apharmaceutically acceptable carrier.

In some embodiments of the methods, the subject has or suspected ofhaving an Alpha 1-Antitrypsin deficiency. In certain embodiments, thesubject has a mutation in an AAT gene. In certain embodiments, themutation encodes a mutant AAT protein. In some embodiments, the methodsfurther comprise determining that the subject has the mutation. Incertain embodiments, the mutation is a mutation listed in Table 1. Incertain embodiments, the mutation is a missense mutation. In certainembodiments, the mutation results in a glutamate to lysine substitutionat amino acid position 366 according to the amino acid sequence setforth as SEQ ID NO: 3. In certain embodiments, the mutant AAT proteinfails to fold properly.

In some embodiments of the methods, the effective amount of rAAV is10¹⁰, 10¹¹, 10¹², or 10¹³ genome copies. In some embodiments,administering is performed intravascularly, intravenously,intrathecally, intraperatoneally, intramuscularly, subcutaneously orintranasally. In certain embodiments, administering is performed byinjection into the hepatic portal vein.

In some embodiments of the methods, administering is performed ex vivoby isolating cells or tissue from a subject, contacting the cell ortissue with an effective amount of an rAAV, thereby producingtransfected cells or tissue, and administering the transfected cells ortissue to the subject. In certain embodiments, the tissue is adiposetissue. In certain embodiments, the cells are stem cells derived fromadipose tissue. In some embodiments, administering the transfected cellsis performed intravascularly, intravenously, intrathecally,intraperatoneally, intramuscularly, subcutaneously or intranasally. Incertain embodiments, administering the transfected cells is performed bytransplantation of transfected cells into a target tissue. In certainembodiments, the target tissue is lung or liver

In some embodiments of the methods, the subject is a mouse, a rat, arabbit, a dog, a cat, a sheep, a pig, a non-human primate or a human. Incertain embodiments, the subject is a human.

In some embodiments of the methods, after administration of the rAAV thelevel of expression of the first protein is determined in the subject.In some embodiments, after administration of the rAAV the level ofexpression of the second protein is determined in the subject. In someembodiments, administering is performed on two or more occasions. Incertain embodiments, the level of the first protein and/or the level ofthe second protein in the subject are determined after at least oneadministration.

In some embodiments of the methods, the serum level of the first proteinin the subject is reduced by at least 85% following administration ofthe rAAV. In some embodiments, the serum level of the first protein inthe subject is reduced by at least 90% following administration of therAAV. In some embodiments, the serum level of the first protein in thesubject is reduced by at least 95% following administration of the rAAV.In some embodiments, the serum level of the first protein in the subjectis reduced by at least 85% within 2 weeks following administration ofthe rAAV. In some embodiments, the serum level of the first protein inthe subject is reduced by at least 90% within 2 weeks followingadministration of the rAAV. In some embodiments, the serum level of thefirst protein in the subject is reduced by at least 85% within 4 weeksof administration of the rAAV. In some embodiments, after 7 weeks ofadministration of the rAAV, the serum level of the first protein is at alevel of at least 50% compared with the serum level of the first proteinprior to administration of the rAAV. In some embodiments, after 7 weeksof administration of the rAAV, the serum level of the first protein isat a level of at least 75% compared with the serum level of the firstprotein prior to administration of the rAAV.

In some embodiments of the methods, after administration of the rAAV atleast one clinical outcome parameter associated with the AAT deficiencyis evaluated in the subject. In some embodiments, the at least oneclinical outcome parameter evaluated after administration of the rAAV iscompared with the at least one clinical outcome parameter determinedprior to administration of the rAAV to determine effectiveness of therAAV, wherein an improvement in the clinical outcome parameter afteradministration of the rAAV indicates effectiveness of the rAAV. In someembodiments, the clinical outcome parameter is selected from the groupconsisting of: serum levels of the first protein, serum levels of thesecond protein, presence of intracellular AAT globules, presence ofinflammatory foci, breathing capacity, cough frequency, phlegmproduction, frequency of chest colds or pneumonia, and tolerance forexercise. In some embodiments, the intracellular AAT globules orinflammatory foci are evaluated in lung tissue or liver tissue.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B Comparison of shRNA and miRNA mediated knockdown of humanAAT. HEK-293 cells were contrasfected with human Z-AAT plasmid andeither a plasmid expressing 3 anti-AAT shRNAs from a U6 promoter or aplasmid expressing 3 anti-AAT miRNA from a hybrid chicken beta actinpromoter. (FIG. 1A) Culture media was harvested at 24, 48 and 72 hoursand was analyzed for the AAT concentration by ELISA. (FIG. 1B) At 72hours cells were harvested and lysed for AAT concentration by ELISA.*<0.05 as determined by a two-way unpaired student t-test.

FIG. 2 In vivo silencing of human AAT by rAAV9 expressed miRNAs.Transgenic mice expressing the human PiZ allele were injected with5×10¹¹ vector particles or rAAV9 expressing miRNAs against AAT under thecontrol of the hybrid chicken beta-actin promoter via the tail vein.Serums from each cohort were collected on a weekly basis and were usedto assess Z-AAT concentration by ELISA. Data is expressed as groupmeans+SEM (n=6).

FIGS. 3A-3G Liver histology for PiZ transgenic mice 5 weeks post-rAAV9delivery. Livers from mice receiving rAAV9 vectors with miRNAs and GFPcontrols were formalin-fixed and stained for AAT, or with a PAS-D assay.Mouse liver sections stained using an anti-human AAT antibody from amouse treated with (FIG. 3A) intronic-3×miR or (FIG. 3B) GFP controls.Mouse Liver sections stained with diastase-resistant Periodic AcidSchiff assay from (FIGS. 3E and 3F) intronic-3×miR or (FIGS. 3C and 3D)GFP controls. (FIG. 3G) Quantitative pixel image analysis of whole liversections was performed by comparing pixel counts of PASD-positiveglobules in GFP controls (N=7) to pixel counts of PASD-positive globulesin intronic-3×miR (N=7).

FIG. 4 In vivo optimization of anti-AAT miRNA delivery within rAAV9vectors. Transgenic mice expressing the human PiZ allele were injectedwith 5×10¹¹ vector particles or rAAV9 expressing miRNAs against AATunder the control of the hybrid chicken beta-actin promoter via the tailvein. Serums from each cohort were collected on a weekly basis and wereused to assess Z-AAT concentration by ELISA.

FIG. 5 Quantitative RT-PCR for artificial miRNA in vivo. Total RNA frommouse livers was used to assay for the presence of the 3 artificialanti-AAT miRNAs from mice receiving rAAV9-miRNA vectors. *<0.05 asdetermined by a two-way unpaired student t-test.

FIGS. 6A-6F Long-term In vivo silencing of human AAT by rAAV9 expressedmiRNAs. Transgenic mice expressing the human PiZ allele were injectedwith 1×1012 vector particles or rAAV9 expressing miRNAs against AATunder the control of the hybrid chicken beta-actin promoter via the tailvein. (FIG. 6A) Serums from each cohort were collected on a weekly basisand were used to assess Z-AAT concentration by ELISA. (FIG. 6B) ATT fromliver lysates of mice was analyzed by immunoblot after monomer andpolymer separation. The 52 kDa Z-AAT was from livers processed andseparated into a monomer and polymer pool. Densitometric analysis wasperformed for the (FIG. 6C) monomer and (FIG. 6D) polymer pools usingImage J software. Baseline serums and those collected two weeks-postrAAV9 delivery were used to analyze liver function as determined by(FIG. 6E) ALT and (FIG. 6F) AST concentration. Data is expressed asgroup means+SEM. *<0.05 as determined by a two-way unpaired student¬t-test comparing rAAV9 cohorts vs. baseline.

FIGS. 7A-7B In vitro assessment of dual-function pro-viral plasmid.HEK-293 cells were contrasfected with human Z-AAT plasmid and either theDouble-6×miR-CB-AAT plasmid, a GFP or PBS control. Cells were processedfor RNA at 72 hours and were analyzed for (FIG. 7A) PiZ-mRNA or (FIG.7B) PiM mRNA with qRT-PCR. Data is expressed as group means+SEM (n=6).*<0.05 as determined by a two-way unpaired student t-test.

FIGS. 8A-8C In vivo knockdown of Z-AAT with simultaneous augmentation ofM-AAT after rAAV9 dual function vector delivery. Transgenic miceexpressing the human PiZ allele were injected with 1×10¹² vectorparticles or rAAV9 expressing miRNAs against AAT and a de-targeted cMyctagged wiltype M-AAT cDNA under the control of the hybrid chickenbeta-actin promoter via the tail vein. (FIG. 8A) Serum from each cohortwas collected on a weekly basis and was used to assess Z-AATconcentration by Z-AAT specific ELISA and M-AAT levels by cMyc ELISA.Total RNA from mouse livers was used to assay for the presence of theeither (FIG. 8) Z-AAT mRNA or (FIG. 8C) M-AAT mRNA by qRT-PCR. Data isexpressed as group means+SEM (n=6). *<0.05 as determined by a two-wayunpaired student t-test.

FIG. 9 Artificial miRNA have minimal impact on endogenous miRNA liverprofiles. Liver RNA was harvested 3 months post delivery from animalsinjected with the following vectors: intronic-3×miR-GFP,PolyA-3×miR-GFP, Double-6×miR-GFP, CB-GFP along with RNA form untreatedPiZ mice and wiltype C57Bl6 mice was used to run a miRNA microarray.Each group consisted of 5 mouse RNA samples and was run independentlywith a single color (Cy5) microarray.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Aspects of the invention relate to improved gene therapy compositionsand related methods for treating Alpha-1 Antitrypsin (AAT, alsosometimes called SERPINA1) deficiencies using the recombinantadeno-associated viral (rAAV) vectors. In some embodiments, a non-toxicdual function vector is provided that is capable of knocking-down mutantAAT while expressing wild-type AAT. The rAAV-based vectors and relatedmethods provide for long-term expression of therapeutic miRNAs andexpression of wild-type protein. According to other aspects, rAAV-basedcompositions and methods are provided that simultaneously directsilencing agents to the liver to decrease Z-AAT expression and directgene expression to other sites (e.g., lung tissue). In some embodiments,compositions and methods are provided that are useful for treating theAAT deficiency by knocking down PiZ protein (a mutant AAT protein) whileat the same time increasing levels of the M-AAT protein (the wild-typeAAT protein). It will be appreciated that the rAAV-based therapeuticapproaches disclosed herein can be applied to other gain-of-function ordominant-negative genetic disorders such as Huntington's disease, whichpreviously have not been amiable to a single vector gene therapyapproach.

Certain rAAV vectors provided herein incorporate miRNA sequencestargeting the AAT gene while driving the expression of hardenedwild-type AAT gene (a wild-type AAT gene that is not targeted by themiRNA), thus achieving concomitant mutant AAT knockdown e.g., in theliver, with increased expression of wildtype AAT. In one embodiment,transgenic mice expressing the human PiZ allele were injected withcontrol or dual function rAAV9 vectors expressing both miRNAs and ahardened AAT gene with a cMyc tag. In this embodiment, serum PiZ levelswere consistently knocked down by an average of 80% from baseline levelswith the knockdown being stable and persistent over a 13 week period. Inone embodiment, cohorts receiving dual function vectors exhibitedknockdown of PiZ AAT while secreting increased serum levels of wild-typeAAT as determined by a PiZ and PiM specific ELISAs. In this embodiment,liver histology revealed significantly decreased globular accumulationof misfolded PiZ AAT in hepatocytes along with a reduction ininflammatory infiltrates when compared to controls.

In one embodiment, global miRNA expression profiles of the liver wereminimally affected by artificial miRNAs delivered via rAAV, with only afew miRNAs showing statistically significant differences. In oneembodiment, a difference was seen in miR-1 which was reduced in PiZtransgenic mice receiving rAAV vectors to normal levels seen inwild-type B6 mice. In one embodiment, the levels of miR-122 wereunaffected in all mice receiving rAAVs expressing miRNA targeting theAAT gene. Accordingly, in some embodiments, dual function rAAV vectorsare effective at knocking down PIZ AAT while simultaneously augmentingwild-type AAT without disturbing endogenous miRNA liver profiles.

Alpha-1 Antitrypsin Deficiency

Alpha-1 antitrypsin (AAT), also known in the art as serpin peptidaseinhibitor, clade A (SERPINA1), is a protein that functions as proteinase(protease) inhibitor. AAT is mainly produced in the liver, but functionsin the lungs and liver, primarily. As used herein the term, “alpha-1antitrypsin deficiency” refers to a condition resulting from adeficiency of functional AAT in a subject. In some embodiments, asubject having an AAT deficiency produces insufficient amounts ofalpha-1 antitrypsin. In some embodiments, a subject having an AATdeficiency produces a mutant AAT. In some embodiments, insufficientamounts of AAT or expression of mutant AAT results in damage to asubject's lung and/or liver. In some embodiments, the AAT deficiencyleads to emphysema and/or liver disease. Typically, AAT deficienciesresult from one or more genetic defects in the AAT gene. The one or moredefects may be present in one or more copies (e.g., alleles) of the AATgene in a subject. Typically, AAT deficiencies are most common amongEuropeans and North Americans of European descent. However, AATdeficiencies may be found in subjects of other descents as well.

Subjects (e.g., adult subjects) with severe AAT deficiencies are likelyto develop emphysema. Onset of emphysema often occurs before age 40 inhuman subjects having AAT deficiencies. Smoking can increase the risk ofemphysema in subjects having AAT deficiencies. Symptoms of AATdeficiencies include shortness of breath, with and without exertion, andother symptoms commonly associated with chronic obstructive pulmonarydisease (COPD). Other symptoms of AAT deficiencies include symptoms ofsevere liver disease (e.g., cirrhosis), unintentional weight loss, andwheezing. A physical examination may reveal a barrel-shaped chest,wheezing, or decreased breath sounds in a subject who has an AATdeficiency.

The following exemplary tests may assist with diagnosing a subject ashaving an AAT deficiency: an alpha-1 antitrypsin blood test, examinationof arterial blood gases, a chest x-ray, a CT scan of the chest, genetictesting, and lung function test. In some cases, a subject having orsuspected of having an AAT deficiency is subjected to genetic testing todetect the presence of one or more mutations in the AAT gene. In someembodiments, one or more of the mutations listed in Table 1 are detectedin the subject.

In some cases, a physician may suspect that a subject has an AATdeficiency if the subject has emphysema at an early age (e.g., beforethe age of 45), emphysema without ever having smoked or without everhaving been exposed to toxins, emphysema with a family history of an AATdeficiency, liver disease or hepatitis when no other cause can be found,liver disease or hepatitis and a family history of an AAT deficiency.

In some embodiments, alpha-1 antitrypsin deficiency can result in twodistinct pathologic states: a lung disease which is primarily due to theloss of anti-protease function, and a liver disease due to a toxic gainof function of the mutant AAT protein (e.g., mutant PiZ-AAT). Forexample, since mutant AAT-PiZ exhibits a gain-of-function hepatocellulartoxicity accumulating in the endoplasmic reticulum, therapies aimed atdecreasing AAT-PiZ mRNA levels may ameliorate or even reverse the liverpathology. In addition, increased secretion of functional AAT proteinprotects the lungs from neutrophil elastase and associated proteolyticenzymes. Applicants have developed several rAAV vectors that provide fordelivery of microRNAs targeted against mutant AAT, within the sameproviral cassette as a gene encoding wild-type AAT. In some embodiments,the microRNAs are delivered using rAAV vectors that have previously beenused in clinical trials.

Isolated Nucleic Acids

In general, the invention provides isolated nucleic acids, which may berAAV vectors, useful for treating genetic disease. The isolated nucleicacids typically comprise one or more regions that encode one or moreinhibitory RNAs that target an endogenous mRNA of a subject. Theisolated nucleic acids also typically comprise one or more regions thatencode one or more exogenous mRNAs. The protein(s) encoded by the one ormore exogenous mRNAs may or may not be different in sequence compositionthan the protein(s) encoded by the one or more endogenous mRNAs. Forexample, the one or more endogenous mRNAs may encode a wild-type andmutant version of a particular protein, such as may be the case when asubject is heterozygous for a particular mutation, and the exogenousmRNA may encode a wild-type mRNA of the same particular protein. In thiscase, typically the sequence of the exogenous mRNA and endogenous mRNAencoding the wild-type protein are sufficiently different such that theexogenous mRNA is not targeted by the one or more inhibitory RNAs. Thismay be accomplished, for example, by introducing one or more silentmutations into the exogenous mRNA such that it encodes the same proteinas the endogenous mRNA but has a different nucleic acid sequence. Inthis case, the exogenous mRNA may be referred to as “hardened.”Alternatively, the inhibitory RNA (e.g. miRNA) can target the 5′ and/or3′ untranslated regions of the endogenous mRNA. These 5′ and/or 3′regions can then be removed or replaced in the exogenous mRNA such thatthe exogenous mRNA is not targeted by the one or more inhibitory RNAs.

In another example, the one or more endogenous mRNAs may encode onlymutant versions of a particular protein, such as may be the case when asubject is homozygous for a particular mutation, and the exogenous mRNAmay encode a wild-type mRNA of the same particular protein. In thiscase, the sequence of the exogenous mRNA may be hardened as describedabove, or the one or more inhibitory RNAs may be designed todiscriminate the mutated endogenous mRNA from the exogenous mRNA.

In some cases, the isolated nucleic acids typically comprise a firstregion that encodes one or more first inhibitory RNAs (e.g., miRNAs)comprising a nucleic acid having sufficient sequence complementary withan endogenous mRNA of a subject to hybridize with and inhibit expressionof the endogenous mRNA, in which the endogenous mRNA encodes a firstprotein. The isolated nucleic acids also typically include a secondregion encoding an exogenous mRNA that encodes a second protein, inwhich the second protein has an amino acid sequence that is at least 85%identical to the first protein, in which the one or more firstinhibitory RNAs do not comprise a nucleic acid having sufficientsequence complementary to hybridize with and inhibit expression of theexogenous mRNA. For example, the first region may be positioned at anysuitable location. The first region may be positioned within anuntranslated portion of the second region. The first region may bepositioned in any untranslated portion of the nucleic acid, including,for example, an intron, a 5′ or 3′ untranslated region, etc.

In some cases, it may be desirable to position the first region upstreamof the first codon of the exogenous mRNA. For example, the first regionmay be positioned between the first codon of the exogenous mRNA and 2000nucleotides upstream of the first codon. The first region may bepositioned between the first codon of the exogenous mRNA and 1000nucleotides upstream of the first codon. The first region may bepositioned between the first codon of the exogenous mRNA and 500nucleotides upstream of the first codon. The first region may bepositioned between the first codon of the exogenous mRNA and 250nucleotides upstream of the first codon. The first region may bepositioned between the first codon of the exogenous mRNA and 150nucleotides upstream of the first codon.

In some cases, the first region may be positioned downstream of aportion of the second region encoding the poly-A tail of the exogenousmRNA. The first region may be between the last codon of the exogenousmRNA and a position 2000 nucleotides downstream of the last codon. Thefirst region may be between the last codon of the exogenous mRNA and aposition 1000 nucleotides downstream of the last codon. The first regionmay be between the last codon of the exogenous mRNA and a position 500nucleotides downstream of the last codon. The first region may bebetween the last codon of the exogenous mRNA and a position 250nucleotides downstream of the last codon. The first region may bebetween the last codon of the exogenous mRNA and a position 150nucleotides downstream of the last codon.

The nucleic acid may also comprise a third region encoding a one or moresecond inhibitory RNAs (e.g., miRNAs) comprising a nucleic acid havingsufficient sequence complementary to hybridize with and inhibitexpression of the endogenous mRNA. As with the first region, the thirdregion may be positioned at any suitable location. For example, thethird region may be positioned in an untranslated portion of the secondregion, including, for example, an intron, a 5′ or 3′ untranslatedregion, etc. The third region may be positioned upstream of a portion ofthe second region encoding the first codon of the exogenous mRNA. Thethird region may be positioned downstream of a portion of the secondregion encoding the poly-A tail of the exogenous mRNA. In some cases,when the first region is positioned upstream of the first codon, thethird region is positioned downstream of the portion of the secondregion encoding the poly-A tail of the exogenous mRNA, and vice versa.

In some cases, the first region and third regions encode the same set ofone or more inhibitory RNAs (e.g., miRNAs). In other cases, the firstregion and third regions encode a different set of one or moreinhibitory RNAs (e.g., miRNAs). In some cases, the one or moreinhibitory RNAs (e.g., miRNAs) encoded by the first region target one ormore of the same genes as the one or more inhibitory RNAs (e.g., miRNAs)encoded by the third region. In some cases, the one or more inhibitoryRNAs (e.g., miRNAs) encoded by the first region do not target any of thesame genes as the one or more inhibitory RNAs (e.g., miRNAs) encoded bythe third region. It is to be appreciated that inhibitory RNAs (e.g.,miRNAs) which target a gene have sufficient complementarity with thegene to bind to and inhibit expression (e.g., by degradation orinhibition of translation) of the corresponding mRNA.

The first and third regions may also encode a different number ofinhibitory RNAs (e.g., miRNAs). For example, the first region and thirdregions may independently encode 1, 2, 3, 4, 5, 6 or more inhibitoryRNAs (e.g., miRNAs). The first and third regions are not limited tocomprising any one particular inhibitory RNA, and may include, forexample, a miRNA, an shRNA, a TuD RNA, a microRNA sponge, an antisenseRNA, a ribozyme, an aptamer, or other appropriate inhibitory RNA. Insome cases, the first region and/or third region comprises one or moremiRNAs. The one or more miRNAs may comprise a nucleic acid sequenceencoded by a sequence selected from the group consisting of SEQ ID NOS:17-19 and 21-23.

As disclosed herein, the second protein may have an amino acid sequencethat is at least 85% identical to the first protein. Accordingly, thesecond protein may have an amino acid sequence that is at least 88%, atleast 90%, at least 95%, at least 98%, at least 99% or more identical tothe first protein. In some case, the second protein differs from thefirst protein by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Insome cases, one or more of the differences between the first protein andsecond protein are conservative amino acid substitutions. As usedherein, a “conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references that compile such methods. Conservativesubstitutions of amino acids include substitutions made among aminoacids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K,R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Accordingly,conservative amino acid substitutions may provide functionallyequivalent variants, or homologs of an endogenous protein.

It should be appreciated that in some cases the second protein may be amarker protein (e.g., a fluorescent protein, a fusion protein, a taggedprotein, etc.). Such constructs may be useful, for example, for studyingthe distribution of the encoded proteins within a cell or within asubject and are also useful for evaluating the efficiency of rAAVtargeting and distribution in a subject.

In some embodiments of the isolated nucleic acids, the first protein isalpha-1 antitrypsin (AAT) protein. An exemplary sequence of a wild-typeAAT is provided at SEQ ID NO: 1 or 2. Accordingly, in some cases, theendogenous mRNA may comprise the RNA sequence specified by the sequenceset forth in SEQ ID NO: 5. The endogenous mRNA may comprise the RNAsequence as specified by any one of the sequences set forth in SEQ IDNOS: 6-16. In some cases, the AAT protein is a human AAT protein. TheAAT protein may have a sequence as set forth in SEQ ID NO: 1 or 2 or oneor more mutations thereof as identified in Table 1, e.g. SEQ ID NO: 3 or4. The exogenous mRNA may have one or more silent mutations comparedwith the endogenous mRNA. The exogenous mRNA may comprise the RNAsequence specified by the sequence set forth in SEQ ID NO: 20. Theexogenous mRNA sequence may or may not encode a peptide tag (e.g., a myctag, a his-tag, etc.) linked to the encoded protein. Often, in aconstruct used for clinical purposes, the exogenous mRNA sequence doesnot encode a peptide tag linked to the encoded protein.

As described further below, the isolated nucleic acids may compriseinverted terminal repeats (ITR) of an AAV serotypes selected from thegroup consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9,AAV10, AAV11 and variants thereof. The isolated nucleic acids may alsoinclude a promoter operably linked with the one or more first inhibitoryRNAs, the exogenous mRNA, and/or the one or more second inhibitory RNAs.The promoter may be tissue-specific promoter, a constitutive promoter orinducible promoter.

TABLE 1 Mutations in Human AAT - Entrez Gene ID: 5265 Amino Chr. mRNAdbSNP rs# dbSNP Protein Codon acid position position cluster id Functionallele residue position position 94844794 1822 rs78787657 missense A Lys[K] 1 417 contig reference C Gln [Q] 1 417 94844797 1819 rs3191200missense C Pro [P] 1 416 contig reference A Thr [T] 1 416 94844842 1774rs17850837 missense A Lys [K] 1 401 contig reference C Gln [Q] 1 40194844843 1773 rs1303 missense C Asp [D] 3 400 contig reference A Glu [E]3 400 94844855 1761 rs13170 synonymous T Phe [F] 3 396 contig referenceC Phe [F] 3 396 94844866 1750 rs61761869 missense T Ser [S] 1 393 contigreference C Pro [P] 1 393 94844887 1729 rs12233 missense T Ser [S] 1 386contig reference C Pro [P] 1 386 94844912 1704 rs28929473 missense T Phe[F] 3 377 contig reference A Leu [L] 3 377 94844926 1690 rs12077missense T Trp [W] 1 373 contig reference G Gly [G] 1 373 94844942 1674rs1050520 synonymous G Lys [K] 3 367 contig reference A Lys [K] 3 36794844947 1669 rs28929474 missense A Lys [K] 1 366 contig reference G Glu[E] 1 366 94844954 1662 rs1050469 synonymous G Thr [T] 3 363 contigreference C Thr [T] 3 363 94844957 1659 rs1802961 synonymous T Leu [L] 3362 contig reference G Leu [L] 3 362 94844959 1657 rs1131154 missense AMet [M] 1 362 contig reference C Leu [L] 1 362 94844960 1656 rs13868synonymous A Val [V] 3 361 contig reference G Val [V] 3 361 948449611655 rs1131139 missense C Ala [A] 2 361 contig reference T Val [V] 2 36194844962 1654 rs72555357 frame shift 1 361 contig reference G Val [V] 1361 94844965 1651 rs1802959 missense A Thr [T] 1 360 contig reference GAla [A] 1 360 94844972 1644 rs10427 synonymous C Val [V] 3 357 contigreference G Val [V] 3 357 94844975 1641 rs9630 synonymous T Ala [A] 3356 contig reference C Ala [A] 3 356 94844977 1639 rs67216923 frameshift 1 356 frame shift (15 bp) 1 356 contig reference G Ala [A] 1 35694845814 1625 rs72555374 frame shift 2 351 contig reference T Leu [L] 2351 94845845 1594 rs28929471 missense A Asn [N] 1 341 contig reference GAsp [D] 1 341 94845893 1546 rs1802962 missense T Cys [C] 1 325 contigreference A Ser [S] 1 325 94845902 1537 rs55704149 missense T Tyr [Y] 1322 contig reference G Asp [D] 1 322 94845914 1525 rs117001071 missenseT Ser [S] 1 318 contig reference A Thr [T] 1 318 94845917 1521rs35624994 frame shift Ser [S] 3 316 frame shift C Ser [S] 3 316 contigreference CA Ser [S] 3 316 94847218 1480 rs1802963 nonsense T xxx [X] 1303 contig reference G Glu [E] 1 303 94847262 1436 rs17580 missense TVal [V] 2 288 contig reference A Glu [E] 2 288 94847285 1413 rs1049800synonymous C Asp [D] 3 280 contig reference T Asp [D] 3 280 948473061392 rs2230075 synonymous T Thr [T] 3 273 contig reference C Thr [T] 3273 94847351 1347 rs34112109 synonymous A Lys [K] 3 258 contig referenceG Lys [K] 3 258 94847357 1341 rs8350 missense G Trp [W] 3 256 contigreference T Cys [C] 3 256 94847386 1312 rs28929470 missense T Cys [C] 1247 contig reference C Arg [R] 1 247 94847407 1291 rs72552401 missense AMet [M] 1 240 contig reference G Val [V] 1 240 94847415 1283 rs6647missense C Ala [A] 2 237 contig reference T Val [V] 2 237 94847452 1246rs11558264 missense C Gln [Q] 1 225 contig reference A Lys [K] 1 22594847466 1232 rs11558257 missense T Ile [I] 2 220 contig reference G Arg[R] 2 220 94847475 1223 rs11558265 missense C Thr [T] 2 217 contigreference A Lys [K] 2 217 94849029 1119 rs113813309 synonymous T Asn [N]3 182 contig reference C Asn [N] 3 182 94849053 1095 rs72552402synonymous T Thr [T] 3 174 contig reference C Thr [T] 3 174 948490611087 rs112030253 missense A Arg [R] 1 172 contig reference G Gly [G] 1172 94849109 1039 rs78640395 nonsense T xxx [X] 1 156 contig reference GGlu [E] 1 156 94849140 1008 rs11558263 missense A Arg [R] 3 145 contigreference C Ser [S] 3 145 94849151 997 rs20546 synonymous T Leu [L] 1142 contig reference C Leu [L] 1 142 94849160 988 rs11558261 missense ASer [S] 1 139 contig reference G Gly [G] 1 139 94849201 947 rs709932missense A His [H] 2 125 contig reference G Arg [R] 2 125 94849228 920rs28931572 missense A Asn [N] 2 116 contig reference T Ile [I] 2 11694849303 845 rs28931568 missense A Glu [E] 2 91 contig reference G Gly[G] 2 91 94849325 823 rs111850950 missense A Thr [T] 1 84 contigreference G Ala [A] 1 84 94849331 817 rs113817720 missense A Thr [T] 182 contig reference G Ala [A] 1 82 94849345 803 rs55819880 missense TPhe [F] 2 77 contig reference C Ser [S] 2 77 94849364 784 rs11575873missense C Arg [R] 1 71 contig reference A Ser [S] 1 71 94849381 767rs28931569 missense C Pro [P] 2 65 contig reference T Leu [L] 2 6594849388 760 rs28931570 missense T Cys [C] 1 63 contig reference C Arg[R] 1 63 94849466 682 rs11558262 missense G Ala [A] 1 37 contigreference A Thr [T] 1 37 94849492 656 rs11558259 missense G Arg [R] 2 28contig reference A Gln [Q] 2 28 94849548 600 rs11558260 synonymous T Ile[I] 3 9 contig reference C Ile [I] 3 9 start codon 1Methods of Use

The invention also provides methods for expressing alpha 1-antitrypsin(AAT) protein in a subject. Typically, the subject has or suspected ofhaving an AAT deficiency. The methods typically involve administering toa subject an effective amount of a recombinant Adeno-Associated Virus(rAAV) harboring any of the isolated nucleic acids disclosed herein. Ingeneral, the “effective amount” of a rAAV refers to an amount sufficientto elicit the desired biological response. In some embodiments, theeffective amount refers to the amount of rAAV effective for transducinga cell or tissue ex vivo. In other embodiments, the effective amountrefers to the amount effective for direct administration of rAAV to asubject. As will be appreciated by those of ordinary skill in this art,the effective amount of the recombinant AAV of the invention variesdepending on such factors as the desired biological endpoint, thepharmacokinetics of the expression products, the condition beingtreated, the mode of administration, and the subject. Typically, therAAV is administered with a pharmaceutically acceptable carrier.

The subject may have a mutation in an AAT gene. The mutation may resultin decreased expression of wild-type (normal) AAT protein. The subjectmay be homozygous for the mutation. The subject may be heterozygous forthe mutation. The mutation may be a missense mutation. The mutation maybe a nonsense mutation. The mutation may be a mutation listed inTable 1. The mutation may result in expression of a mutant AAT protein.The mutant protein may be a gain-of-function mutant or aloss-of-function mutant. The mutant AAT protein may be incapable ofinhibiting protease activity. The mutant AAT protein may fail to foldproperly. The mutant AAT protein may result in the formation of proteinaggregates. The mutant AAT protein may result in the formation ofintracellular AAT globules. The mutation may result in a glutamate tolysine substitution at amino acid position 366 in the precursor proteinaccording to the amino acid sequence set forth as SEQ ID NO: 3. In themature protein, this same mutation occurs at amino acid position 342(SEQ ID NO: 4). The methods may also involve determining whether thesubject has a mutation. Accordingly the methods may involve obtaining agenotype of the AAT gene in the subject.

In some cases, after administration of the rAAV the level of expressionof the first protein and/or second protein is determined in the subject.The administration may be performed on one or more occasions. When theadministration is performed on one or more occasions, the level of thefirst protein and/or the level of the second protein in the subject areoften determined after at least one administration. In some cases, theserum level of the first protein in the subject is reduced by at least85% following administration of the rAAV. The serum level of the firstprotein in the subject may be reduced by at least 90% followingadministration of the rAAV. The serum level of the first protein in thesubject may be reduced by at least 95% following administration of therAAV. However, in some cases, the serum level of the first protein inthe subject is reduced by at least 40%, at least 50%, at least 60%, atleast 70%, or at least 80% following administration of the rAAV.

The level (e.g., serum level) of the first protein in the subject may bereduced by at least 85% within 2 weeks following administration of therAAV. The serum level of the first protein in the subject may be reducedby at least 90% within 2 weeks following administration of the rAAV. Theserum level of the first protein in the subject may be reduced by atleast 85% within 4 weeks of administration of the rAAV. The reductionmay be observed within 1 day, within 2 days, within 3 days, within 4days, within 5 days, within 6 days, within 1 week, within 2 weeks,within 3 weeks, within 4 weeks or more.

The reduction in the level of the first protein may be sustained for atleast 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, atleast 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, atleast 9 weeks, at least 10 weeks, at least 11 weeks, or more. In somecases, after 7 weeks of administration of the rAAV, the serum level ofthe first protein is at a level of at least 50% compared with the serumlevel of the first protein prior to administration of the rAAV. Incertain cases, after 7 weeks of administration of the rAAV, the serumlevel of the first protein is at a level of at least 75% compared withthe serum level of the first protein prior to administration of therAAV.

In some instances, after administration of the rAAV at least oneclinical outcome parameter associated with the AAT deficiency isevaluated in the subject. Typically, the clinical outcome parameterevaluated after administration of the rAAV is compared with the clinicaloutcome parameter determined at a time prior to administration of therAAV to determine effectiveness of the rAAV. Often an improvement in theclinical outcome parameter after administration of the rAAV indicateseffectiveness of the rAAV. Any appropriate clinical outcome parametermay be used. Typically, the clinical outcome parameter is indicative ofthe one or more symptoms of an AAT deficiency. For example, the clinicaloutcome parameter may be selected from the group consisting of: serumlevels of the first protein, serum levels of the second protein,presence of intracellular AAT globules, presence of inflammatory foci,breathing capacity, cough frequency, phlegm production, frequency ofchest colds or pneumonia, and tolerance for exercise. Intracellular AATglobules or inflammatory foci are evaluated in tissues affected by theAAT deficiency, including, for example, lung tissue or liver tissue.

Recombinant AAVs

In some aspects, the invention provides isolated AAVs. As used hereinwith respect to AAVs, the term “isolated” refers to an AAV that has beenisolated from its natural environment (e.g., from a host cell, tissue,or subject) or artificially produced. Isolated AAVs may be producedusing recombinant methods. Such AAVs are referred to herein as“recombinant AAVs”. Recombinant AAVs (rAAVs) preferably havetissue-specific targeting capabilities, such that a transgene of therAAV will be delivered specifically to one or more predeterminedtissue(s). The AAV capsid is an important element in determining thesetissue-specific targeting capabilities. Thus, a rAAV having a capsidappropriate for the tissue being targeted can be selected. In someembodiments, the rAAV comprises a capsid protein having an amino acidsequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, AAV10, AAV11 and variants thereof. The recombinantAAVs typically harbor an isolated nucleic acid of the invention.

Methods for obtaining recombinant AAVs having a desired capsid proteinare well known in the art (See, for example, US 2003/0138772, thecontents of which are incorporated herein by reference in theirentirety). AAV capsid proteins that may be used in the rAAVs of theinvention a include, for example, those disclosed in G. Gao, et al., J.Virol, 78(12):6381-6388 (June 2004); G. Gao, et al, Proc Natl Acad SciUSA, 100(10):6081-6086 (May 13, 2003); US 2003-0138772, US 2007/0036760,US 2009/0197338, and WO 2010/138263, the contents of which relating toAAVs capsid proteins and associated nucleotide and amino acid sequencesare incorporated herein by reference. Typically the methods involveculturing a host cell which contains a nucleic acid sequence encoding anAAV capsid protein or fragment thereof; a functional rep gene; arecombinant AAV vector composed of AAV inverted terminal repeats (ITRs)and a transgene; and sufficient helper functions to permit packaging ofthe recombinant AAV vector into the AAV capsid proteins.

The components to be cultured in the host cell to package a rAAV vectorin an AAV capsid may be provided to the host cell in trans.Alternatively, any one or more of the required components (e.g.,recombinant AAV vector, rep sequences, cap sequences, and/or helperfunctions) may be provided by a stable host cell which has beenengineered to contain one or more of the required components usingmethods known to those of skill in the art. Most suitably, such a stablehost cell will contain the required component(s) under the control of aninducible promoter. However, the required component(s) may be under thecontrol of a constitutive promoter. Examples of suitable inducible andconstitutive promoters are provided herein. In still anotheralternative, a selected stable host cell may contain selectedcomponent(s) under the control of a constitutive promoter and otherselected component(s) under the control of one or more induciblepromoters. For example, a stable host cell may be generated which isderived from 293 cells (which contain E1 helper functions under thecontrol of a constitutive promoter), but which contain the rep and/orcap proteins under the control of inducible promoters. Still otherstable host cells may be generated by one of skill in the art.

The recombinant AAV vector, rep sequences, cap sequences, and helperfunctions required for producing the rAAV of the invention may bedelivered to the packaging host cell using any appropriate geneticelement (vector). The selected genetic element may be delivered by anysuitable method, including those described herein. The methods used toconstruct any embodiment of this invention are known to those with skillin nucleic acid manipulation and include genetic engineering,recombinant engineering, and synthetic techniques. See, e.g., Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Similarly, methods of generating rAAV virionsare well known and the selection of a suitable method is not alimitation on the present invention. See, e.g., K. Fisher et al, J.Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

In some embodiments, recombinant AAVs may be produced using the tripletransfection method (e.g., as described in detail in U.S. Pat. No.6,001,650, the contents of which relating to the triple transfectionmethod are incorporated herein by reference). Typically, the recombinantAAVs are produced by transfecting a host cell with a recombinant AAVvector (comprising a transgene) to be packaged into AAV particles, anAAV helper function vector, and an accessory function vector. An AAVhelper function vector encodes the “AAV helper function” sequences(i.e., rep and cap), which function in trans for productive AAVreplication and encapsidation. Preferably, the AAV helper functionvector supports efficient AAV vector production without generating anydetectable wild-type AAV virions (i.e., AAV virions containingfunctional rep and cap genes). Non-limiting examples of vectors suitablefor use with the present invention include pHLP19, described in U.S.Pat. No. 6,001,650 and pRep6cap6 vector, described in U.S. Pat. No.6,156,303, the entirety of both incorporated by reference herein. Theaccessory function vector encodes nucleotide sequences for non-AAVderived viral and/or cellular functions upon which AAV is dependent forreplication (i.e., “accessory functions”). The accessory functionsinclude those functions required for AAV replication, including, withoutlimitation, those moieties involved in activation of AAV genetranscription, stage specific AAV mRNA splicing, AAV DNA replication,synthesis of cap expression products, and AAV capsid assembly.Viral-based accessory functions can be derived from any of the knownhelper viruses such as adenovirus, herpesvirus (other than herpessimplex virus type-1), and vaccinia virus.

In some aspects, the invention provides transfected host cells. The term“transfection” is used to refer to the uptake of foreign DNA by a cell,and a cell has been “transfected” when exogenous DNA has been introducedinside the cell membrane. A number of transfection techniques aregenerally known in the art. See, e.g., Graham et al. (1973) Virology,52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual,Cold Spring Harbor Laboratories, New York, Davis et al. (1986) BasicMethods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene13:197. Such techniques can be used to introduce one or more exogenousnucleic acids, such as a nucleotide integration vector and other nucleicacid molecules, into suitable host cells.

A “host cell” refers to any cell that harbors, or is capable ofharboring, a substance of interest. Often a host cell is a mammaliancell. A host cell may be used as a recipient of an AAV helper construct,an AAV minigene plasmid, an accessory function vector, or other transferDNA associated with the production of recombinant AAVs. The termincludes the progeny of the original cell which has been transfected.Thus, a “host cell” as used herein may refer to a cell which has beentransfected with an exogenous DNA sequence. It is understood that theprogeny of a single parental cell may not necessarily be completelyidentical in morphology or in genomic or total DNA complement as theoriginal parent, due to natural, accidental, or deliberate mutation.

In some aspects, the invention provides isolated cells. As used hereinwith respect to cell, the term “isolated” refers to a cell that has beenisolated from its natural environment (e.g., from a tissue or subject).As used herein, the term “cell line” refers to a population of cellscapable of continuous or prolonged growth and division in vitro. Often,cell lines are clonal populations derived from a single progenitor cell.It is further known in the art that spontaneous or induced changes canoccur in karyotype during storage or transfer of such clonalpopulations. Therefore, cells derived from the cell line referred to maynot be precisely identical to the ancestral cells or cultures, and thecell line referred to includes such variants. As used herein, the terms“recombinant cell” refers to a cell into which an exogenous DNA segment,such as DNA segment that leads to the transcription of abiologically-active polypeptide or production of a biologically activenucleic acid such as an RNA, has been introduced.

As used herein, the term “vector” includes any genetic element, such asa plasmid, phage, transposon, cosmid, chromosome, artificial chromosome,virus, virion, etc., which is capable of replication when associatedwith the proper control elements and which can transfer gene sequencesbetween cells. Thus, the term includes cloning and expression vehicles,as well as viral vectors. In some embodiments, useful vectors arecontemplated to be those vectors in which the nucleic acid segment to betranscribed is positioned under the transcriptional control of apromoter. A “promoter” refers to a DNA sequence recognized by thesynthetic machinery of the cell, or introduced synthetic machinery,required to initiate the specific transcription of a gene. The phrases“operatively positioned,” “under control” or “under transcriptionalcontrol” means that the promoter is in the correct location andorientation in relation to the nucleic acid to control RNA polymeraseinitiation and expression of the gene. The term “expression vector orconstruct” means any type of genetic construct containing a nucleic acidin which part or all of the nucleic acid encoding sequence is capable ofbeing transcribed. In some embodiments, expression includestranscription of the nucleic acid, for example, to generate abiologically-active polypeptide product or inhibitory RNA (e.g., shRNA,miRNA) from a transcribed gene.

The foregoing methods for packaging recombinant vectors in desired AAVcapsids to produce the rAAVs of the invention are not meant to belimiting and other suitable methods will be apparent to the skilledartisan.

Recombinant AAV Vectors

The isolated nucleic acids of the invention may be recombinant AAVvectors. The recombinant AAV vector may be packaged into a capsidprotein and administered to a subject and/or delivered to a selectedtarget cell. “Recombinant AAV (rAAV) vectors” are typically composed of,at a minimum, a transgene and its regulatory sequences, and 5′ and 3′AAV inverted terminal repeats (ITRs). The transgene may comprise, asdisclosed elsewhere herein, one or more regions that encode one or moreinhibitory RNAs (e.g., miRNAs) comprising a nucleic acid that targets anendogenous mRNA of a subject. The transgene may also comprise a regionencoding an exogenous mRNA that encodes a protein (e.g., a protein thathas an amino acid sequence that is at least 85% identical to the proteinencoded by the endogenous mRNA), in which the one or more inhibitoryRNAs do not target the exogenous mRNA.

The AAV sequences of the vector typically comprise the cis-acting 5′ and3′ inverted terminal repeat sequences (See, e.g., B. J. Carter, in“Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168(1990)). The ITR sequences are about 145 bp in length. Preferably,substantially the entire sequences encoding the ITRs are used in themolecule, although some degree of minor modification of these sequencesis permissible. The ability to modify these ITR sequences is within theskill of the art. (See, e.g., texts such as Sambrook et al, “MolecularCloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory,New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). Anexample of such a molecule employed in the present invention is a“cis-acting” plasmid containing the transgene, in which the selectedtransgene sequence and associated regulatory elements are flanked by the5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained fromany known AAV, including presently identified mammalian AAV types.

In addition to the major elements identified above for the recombinantAAV vector, the vector also includes conventional control elements whichare operably linked with elements of the transgene in a manner thatpermits its transcription, translation and/or expression in a celltransfected with the vector or infected with the virus produced by theinvention. As used herein, “operably linked” sequences include bothexpression control sequences that are contiguous with the gene ofinterest and expression control sequences that act in trans or at adistance to control the gene of interest. Expression control sequencesinclude appropriate transcription initiation, termination, promoter andenhancer sequences; efficient RNA processing signals such as splicingand polyadenylation (polyA) signals; sequences that stabilizecytoplasmic mRNA; sequences that enhance translation efficiency (i.e.,Kozak consensus sequence); sequences that enhance protein stability; andwhen desired, sequences that enhance secretion of the encoded product. Anumber of expression control sequences, including promoters which arenative, constitutive, inducible and/or tissue-specific, are known in theart and may be utilized.

As used herein, a nucleic acid sequence (e.g., coding sequence) andregulatory sequences are said to be operably linked when they arecovalently linked in such a way as to place the expression ortranscription of the nucleic acid sequence under the influence orcontrol of the regulatory sequences. If it is desired that the nucleicacid sequences be translated into a functional protein, two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably linked to a nucleic acidsequence if the promoter region were capable of effecting transcriptionof that DNA sequence such that the resulting transcript might betranslated into the desired protein or polypeptide. Similarly two ormore coding regions are operably linked when they are linked in such away that their transcription from a common promoter results in theexpression of two or more proteins having been translated in frame. Insome embodiments, operably linked coding sequences yield a fusionprotein. In some embodiments, operably linked coding sequences yield afunctional RNA (e.g., miRNA).

For nucleic acids encoding proteins, a polyadenylation sequencegenerally is inserted following the transgene sequences and before the3′ AAV ITR sequence. A rAAV construct useful in the present inventionmay also contain an intron, desirably located between thepromoter/enhancer sequence and the transgene. One possible intronsequence is derived from SV-40, and is referred to as the SV-40 T intronsequence. Any intron may be from the β-Actin gene. Another vectorelement that may be used is an internal ribosome entry site (IRES).

The precise nature of the regulatory sequences needed for geneexpression in host cells may vary between species, tissues or celltypes, but shall in general include, as necessary, 5′ non-transcribedand 5′ non-translated sequences involved with the initiation oftranscription and translation respectively, such as a TATA box, cappingsequence, CAAT sequence, enhancer elements, and the like. Especially,such 5′ non-transcribed regulatory sequences will include a promoterregion that includes a promoter sequence for transcriptional control ofthe operably joined gene. Regulatory sequences may also include enhancersequences or upstream activator sequences as desired. The vectors of theinvention may optionally include 5′ leader or signal sequences. Thechoice and design of an appropriate vector is within the ability anddiscretion of one of ordinary skill in the art.

Examples of constitutive promoters include, without limitation, theretroviral Rous sarcoma virus (RSV) LTR promoter (optionally with theRSV enhancer), the cytomegalovirus (CMV) promoter (optionally with theCMV enhancer), the SV40 promoter, and the dihydrofolate reductasepromoter. Inducible promoters allow regulation of gene expression andcan be regulated by exogenously supplied compounds, environmentalfactors such as temperature, or the presence of a specific physiologicalstate, e.g., acute phase, a particular differentiation state of thecell, or in replicating cells only. Inducible promoters and induciblesystems are available from a variety of commercial sources, including,without limitation, Invitrogen, Clontech and Ariad. Many other systemshave been described and can be readily selected by one of skill in theart. Examples of inducible promoters regulated by exogenously suppliedpromoters include the zinc-inducible sheep metallothionine (MT)promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus(MMTV) promoter, the T7 polymerase promoter system, the ecdysone insectpromoter, the tetracycline-repressible system, thetetracycline-inducible system, the RU486-inducible system and therapamycin-inducible system. Still other types of inducible promoterswhich may be useful in this context are those which are regulated by aspecific physiological state, e.g., temperature, acute phase, aparticular differentiation state of the cell, or in replicating cellsonly. In another embodiment, the native promoter, or fragment thereof,for the transgene will be used. In a further embodiment, other nativeexpression control elements, such as enhancer elements, polyadenylationsites or Kozak consensus sequences may also be used to mimic the nativeexpression.

In some embodiments, the regulatory sequences impart tissue-specificgene expression capabilities. In some cases, the tissue-specificregulatory sequences bind tissue-specific transcription factors thatinduce transcription in a tissue specific manner. Such tissue-specificregulatory sequences (e.g., promoters, enhancers, etc.) are well knownin the art. In some embodiments, the promoter is a chicken β-actinpromoter.

In some embodiments, one or more bindings sites for one or more ofmiRNAs are incorporated in a transgene of a rAAV vector, to inhibit theexpression of the transgene in one or more tissues of a subjectharboring the transgenes, e.g., non-liver tissues, non-lung tissues. Theskilled artisan will appreciate that binding sites may be selected tocontrol the expression of a transgene in a tissue specific manner. ThemiRNA target sites in the mRNA may be in the 5′ UTR, the 3′ UTR or inthe coding region. Typically, the target site is in the 3′ UTR of themRNA. Furthermore, the transgene may be designed such that multiplemiRNAs regulate the mRNA by recognizing the same or multiple sites. Thepresence of multiple miRNA binding sites may result in the cooperativeaction of multiple RISCs and provide highly efficient inhibition ofexpression. The target site sequence may comprise a total of 5-100,10-60, or more nucleotides. The target site sequence may comprise atleast 5 nucleotides of the sequence of a target gene binding site.

In some embodiments, the cloning capacity of the recombinant RNA vectormay be limited and a desired coding sequence may involve the completereplacement of the virus's 4.8 kilobase genome. Large genes may,therefore, not be suitable for use in a standard recombinant AAV vector,in some cases. The skilled artisan will appreciate that options areavailable in the art for overcoming a limited coding capacity. Forexample, the AAV ITRs of two genomes can anneal to form head to tailconcatamers, almost doubling the capacity of the vector. Insertion ofsplice sites allows for the removal of the ITRs from the transcript.Other options for overcoming a limited cloning capacity will be apparentto the skilled artisan.

Recombinant AAV Administration

rAAVs are administered in sufficient amounts to transfect the cells of adesired tissue and to provide sufficient levels of gene transfer andexpression without undue adverse effects. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the selected tissue (e.g., livertissue, lung tissue) and administration subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, intracerebrally, orally,intraperitoneally, by inhalation or by another route. Routes ofadministration may be combined, if desired. Delivery of certain rAAVs toa subject may be, for example, by administration into the bloodstream ofthe subject. Administration into the bloodstream may be by injectioninto a vein, an artery, or any other vascular conduit.

In certain circumstances it will be desirable to deliver the rAAV-basedtherapeutic constructs in suitably formulated pharmaceuticalcompositions disclosed herein either subcutaneously,intraopancreatically, intranasally, parenterally, intravenously,intramuscularly, intrathecally, intracerebrally, orally,intraperitoneally, or by inhalation.

It can be appreciated by one skilled in the art that desirableadministration of rAAV-based therapeutic constructs can also include exvivo administration. In some embodiments, ex vivo administrationcomprises (1) isolation of cells or tissue(s) of interest from asubject, (2) contacting the cells or tissue(s) with rAAVs in sufficientamounts to transfect the cells or tissue to provide sufficient levels ofgene transfer and expression without undue adverse effect, and (3)transferring cells or tissue back into the subject. In some embodiments,cells or tissues may be cultured ex vivo for several days before and/orafter transfection.

Cells or tissues can be isolated from a subject by any suitable method.For example, cells or tissues may be isolated by surgery, biopsy (e.g.,biopsy of skin tissue, lung tissue, liver tissue, adipose tissue), orcollection of biological fluids such as blood. In some embodiments,cells are isolated from bone marrow. In some embodiments, cells areisolated from adipose tissue. In some embodiments, cells are isolatedfrom a lipoaspirate. Appropriate methods for isolating cells fromadipose tissue for ex vivo transfection are known in the art. See, e.g.,Kuroda, M., et al., (2011), Journal of Diabetes Investigation, 2:333-340; Kouki Morizono, et al. Human Gene Therapy. January 2003, 14(1):59-66; and Patricia A. Zuk, Viral Transduction of Adipose-Derived StemCells, Methods in Molecular Biology, 1, Volume 702, Adipose-Derived StemCells, Part 4, Pages 345-357.

In some embodiments, the isolated cells comprise stem cells, pluripotentstem cells, lipoaspirate derived stem cells, liver cells (e.g.,hepatocytes), hematopeotic stem cells, mesenchymal stem cells, stromalcells, hematopeotic cells, blood cells, fibroblasts, endothelial cells,epithelial cells, or other suitable cells. In some embodiments, cells tobe transfected are induced pluripotent stem cells prepared from cellsisolated from the subject.

In an embodiment, cells or tissue(s) are transduced at a multiplicity ofinfection (MOI) of at least 10 infectious units (i.u.) of a rAAV percell (for example, 10, 100, 1,000, 5,000, 10,000, 100,000 or more i.u.)or at a functionally equivalent viral copy number. In one embodiment,cells or tissue(s) are transduced at a MOI of 10 to 10,000 i.u. Routesfor transfer of transfected cells or tissue(s) into a subject include,but are not limited to, subcutaneously, intraopancreatically,intranasally, parenterally, intravenously, intravascularly,intramuscularly, intrathecally, intracerebrally, intraperitoneally, orby inhalation. In some embodiments, transfected cells are administeredby hepatic portal vein injection. In some embodiments, transfected cellsare administered intravascularly. Methods for ex vivo administration ofrAAV are well known in the art (see, e.g., Naldini, L. Nature ReviewsGenetics (2011) 12, 301-315, Li, H. et al. Molecular Therapy (2010) 18,1553-1558, and Loiler et al. Gene Therapy (2003) 10, 1551-1558).

Recombinant AAV Compositions

The rAAVs may be delivered to a subject in compositions according to anyappropriate methods known in the art. The rAAV, preferably suspended ina physiologically compatible carrier (e.g., in a composition), may beadministered to a subject, e.g., a human, mouse, rat, cat, dog, sheep,rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, ora non-human primate (e.g., Macaque). The compositions of the inventionmay comprise a rAAV alone, or in combination with one or more otherviruses (e.g., a second rAAV encoding having one or more differenttransgenes).

Suitable carriers may be readily selected by one of skill in the art inview of the indication for which the rAAV is directed. For example, onesuitable carrier includes saline, which may be formulated with a varietyof buffering solutions (e.g., phosphate buffered saline). Otherexemplary carriers include sterile saline, lactose, sucrose, calciumphosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, andwater. Still others will be apparent to the skilled artisan.

Optionally, the compositions of the invention may contain, in additionto the rAAV and carrier(s), other conventional pharmaceuticalingredients, such as preservatives, or chemical stabilizers. Suitableexemplary preservatives include chlorobutanol, potassium sorbate, sorbicacid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin,glycerin, phenol, and parachlorophenol. Suitable chemical stabilizersinclude gelatin and albumin.

The dose of rAAV virions required to achieve a desired effect or“therapeutic effect,” e.g., the units of dose in vector genomes/perkilogram of body weight (vg/kg), will vary based on several factorsincluding, but not limited to: the route of rAAV administration, thelevel of gene or RNA expression required to achieve a therapeuticeffect, the specific disease or disorder being treated, and thestability of the gene or RNA product. One of skill in the art canreadily determine a rAAV virion dose range to treat a subject having aparticular disease or disorder based on the aforementioned factors, aswell as other factors that are well known in the art. An effectiveamount of the rAAV is generally in the range of from about 10 μl toabout 100 ml of solution containing from about 109 to 10¹⁶ genome copiesper subject. Other volumes of solution may be used. The volume used willtypically depend, among other things, on the size of the subject, thedose of the rAAV, and the route of administration. For example, forintravenous administration a volume in range of 10 μl to 100 μl, 100 μlto 1 ml, 1 ml to 10 ml, or more may be used. In some cases, a dosagebetween about 10¹⁰ to 10¹² rAAV genome copies per subject isappropriate. In some embodiments the rAAV is administered at a dose of10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, or 10¹⁵ genome copies per subject. In someembodiments the rAAV is administered at a dose of 10¹⁰, 10¹¹, 10¹²,10¹³, or 10¹⁴ genome copies per kg.

In some embodiments, rAAV compositions are formulated to reduceaggregation of AAV particles in the composition, particularly where highrAAV concentrations are present (e.g., ˜10¹³ GC/ml or more). Methods forreducing aggregation of rAAVs are well known in the art and, include,for example, addition of surfactants, pH adjustment, salt concentrationadjustment, etc. (See, e.g., Wright F R, et al., Molecular Therapy(2005) 12, 171-178, the contents of which are incorporated herein byreference.)

Formulation of pharmaceutically-acceptable excipients and carriersolutions is well-known to those of skill in the art, as is thedevelopment of suitable dosing and treatment regimens for using theparticular compositions described herein in a variety of treatmentregimens. Typically, these formulations may contain at least about 0.1%of the active ingredient or more, although the percentage of the activeingredient(s) may, of course, be varied and may conveniently be betweenabout 1 or 2% and about 70% or 80% or more of the weight or volume ofthe total formulation. Naturally, the amount of active ingredient ineach therapeutically-useful composition may be prepared is such a waythat a suitable dosage will be obtained in any given unit dose of thecompound. Factors such as solubility, bioavailability, biologicalhalf-life, route of administration, product shelf life, as well as otherpharmacological considerations will be contemplated by one skilled inthe art of preparing such pharmaceutical formulations, and as such, avariety of dosages and treatment regimens may be desirable.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. Dispersions may also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. In many cases the form issterile and fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For administration of an injectable aqueous solution, for example, thesolution may be suitably buffered, if necessary, and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art. For example, one dosage may be dissolvedin 1 ml of isotonic NaCl solution and either added to 1000 ml ofhypodermoclysis fluid or injected at the proposed site of infusion, (seefor example, “Remington's Pharmaceutical Sciences” 15th Edition, pages1035-1038 and 1570-1580). Some variation in dosage will necessarilyoccur depending on the condition of the host. The person responsible foradministration will, in any event, determine the appropriate dose forthe individual host.

Sterile injectable solutions are prepared by incorporating the activerAAV in the required amount in the appropriate solvent with various ofthe other ingredients enumerated herein, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The rAAV compositions disclosed herein may also be formulated in aneutral or salt form. Pharmaceutically-acceptable salts, include theacid addition salts (formed with the free amino groups of the protein)and which are formed with inorganic acids such as, for example,hydrochloric or phosphoric acids, or such organic acids as acetic,oxalic, tartaric, mandelic, and the like. Salts formed with the freecarboxyl groups can also be derived from inorganic bases such as, forexample, sodium, potassium, ammonium, calcium, or ferric hydroxides, andsuch organic bases as isopropylamine, trimethylamine, histidine,procaine and the like. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as injectable solutions, drug-releasecapsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Supplementary active ingredients can also be incorporated into thecompositions. The phrase “pharmaceutically-acceptable” refers tomolecular entities and compositions that do not produce an allergic orsimilar untoward reaction when administered to a host.

Delivery vehicles such as liposomes, nanocapsules, microparticles,microspheres, lipid particles, vesicles, and the like, may be used forthe introduction of the compositions of the present invention intosuitable host cells. In particular, the rAAV vector delivered transgenesmay be formulated for delivery either encapsulated in a lipid particle,a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.

Such formulations may be preferred for the introduction ofpharmaceutically acceptable formulations of the nucleic acids or therAAV constructs disclosed herein. The formation and use of liposomes isgenerally known to those of skill in the art. Recently, liposomes weredeveloped with improved serum stability and circulation half-times (U.S.Pat. No. 5,741,516). Further, various methods of liposome and liposomelike preparations as potential drug carriers have been described (U.S.Pat. Nos. 5,567,434; 5,552,157; 5,565,213; 5,738,868 and 5,795,587).

Liposomes have been used successfully with a number of cell types thatare normally resistant to transfection by other procedures. In addition,liposomes are free of the DNA length constraints that are typical ofviral-based delivery systems. Liposomes have been used effectively tointroduce genes, drugs, radiotherapeutic agents, viruses, transcriptionfactors and allosteric effectors into a variety of cultured cell linesand animals. In addition, several successful clinical trails examiningthe effectiveness of liposome-mediated drug delivery have beencompleted.

Liposomes are formed from phospholipids that are dispersed in an aqueousmedium and spontaneously form multilamellar concentric bilayer vesicles(also termed multilamellar vesicles (MLVs). MLVs generally havediameters of from 25 nm to 4 μm. Sonication of MLVs results in theformation of small unilamellar vesicles (SUVs) with diameters in therange of 200 to 500 .ANG., containing an aqueous solution in the core.

Alternatively, nanocapsule formulations of the rAAV may be used.Nanocapsules can generally entrap substances in a stable andreproducible way. To avoid side effects due to intracellular polymericoverloading, such ultrafine particles (sized around 0.1 μm) should bedesigned using polymers able to be degraded in vivo. Biodegradablepolyalkyl-cyanoacrylate nanoparticles that meet these requirements arecontemplated for use.

In addition to the methods of delivery described above, the followingtechniques are also contemplated as alternative methods of deliveringthe rAAV compositions to a host. Sonophoresis (ie., ultrasound) has beenused and described in U.S. Pat. No. 5,656,016 as a device for enhancingthe rate and efficacy of drug permeation into and through thecirculatory system. Other drug delivery alternatives contemplated areintraosseous injection (U.S. Pat. No. 5,779,708), microchip devices(U.S. Pat. No. 5,797,898), ophthalmic formulations (Bourlais et al.,1998), transdermal matrices (U.S. Pat. Nos. 5,770,219 and 5,783,208) andfeedback-controlled delivery (U.S. Pat. No. 5,697,899).

Kits and Related Compositions

The isolated nucleic acids, compositions, rAAV vectors, rAAVs, etc.described herein may, in some embodiments, be assembled intopharmaceutical or diagnostic or research kits to facilitate their use intherapeutic, diagnostic or research applications. A kit may include oneor more containers housing the components of the invention andinstructions for use. Specifically, such kits may include one or moreagents described herein, along with instructions describing the intendedapplication and the proper use of these agents. In certain embodimentsagents in a kit may be in a pharmaceutical formulation and dosagesuitable for a particular application and for a method of administrationof the agents. Kits for research purposes may contain the components inappropriate concentrations or quantities for running variousexperiments.

The kit may be designed to facilitate use of the methods describedherein by researchers and can take many forms. Each of the compositionsof the kit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. As used herein,“instructions” can define a component of instruction and/or promotion,and typically involve written instructions on or associated withpackaging of the invention. Instructions also can include any oral orelectronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for animal administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a subject. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to asubject, such as a syringe, topical application devices, or IV needletubing and bag.

Exemplary embodiments of the invention will be described in more detailby the following examples. These embodiments are exemplary of theinvention, which one skilled in the art will recognize is not limited tothe exemplary embodiments.

EXAMPLES

Introduction to the Examples

Alpha-1 antitrypsin (AAT) deficiency is one of the most commonlyinherited diseases in North America, with a carrier frequency ofapproximately 4% in the US population. The most common mutation arisesas a single base pair change (Glu342Lys, PI*Z, SEQ ID 4) and leads tothe synthesis of the mutant Z-AAT protein, which polymerizes andaccumulates within hepatocytes, precluding its efficient secretion. Thesubsequent relative deficiency of serum AAT predisposes to chronic lungdisease. Twelve to 15% of homozygous PI*ZZ patients develop significantliver disease, ranging from neonatal hepatitis, cholestatic jaundice andcirrhosis to adult-onset cirrhosis and hepatocellular carcinoma. Liverinjury is considered to be a consequence of the pathologicalaccumulation of mutant Z-AAT protein polymers within the endoplasmicreticulum of hepatocytes.

Strategies to alleviate the liver disease are focused on decreasing thepresence of the mutant Z-AAT protein in the hepatocytes by eitherreducing expression of the mutant protein, or augmenting its proteolysisor secretion. In vivo studies of an allele-specific small interferingRNA (siRNA) directed against PI*Z AAT in the Pi*Z transgenic mouse modelof AAT deficiency have been performed. In vitro studies using U6-drivenshRNA clones in recombinant adeno-associated virus (rAAV) backbones haveidentified an effective allele-specific siRNA sequence (termed p10) thatcan reduce Pi*Z AAT protein levels while minimizing knockdown of thenormal Pi*M AAT. Using the AAV8 capsid, rAAV-U6-p10 was packaged andadministered by hepatic portal vein injection into Pi*Z transgenic micefor direct in vivo targeting of the liver. A similarly deliveredAAV8-packaged non-specific siRNA, rAAV-U6-NC, served as a control (NC).Histological data from these studies revealed areas of complete orpartial elimination of Z-AAT protein in the liver at 10 dayspost-injection in the p10 cohort. Analysis of the serum Z-AAT levelsshows a kinetically significant reduction for 4 weeks post-injection inthe p10 cohort when compared to NC control cohort. To examine theallele-specificity, AAV8-packaged Pi*M-AAT was co-administered with eachshRNA construct. For both the p10+Pi*M and NC+Pi*M groups, there wasconsiderable expression of AAT in the liver by histological staining andthere was no significant difference in serum AAT levels.

The Pi*Z mutation (Glu342Lys) within exon 5 of alpha-1 antitrypsin (AAT)causes a plasma AAT deficiency (A1AD) which exposes lung tissue touncontrolled proteolytic attack and can result in emphysema. Pi*Z mutantAAT is retained within the hepatocytes and causes a liver disease in˜12% of patients with the deficiency. Delivering wild-type copies of AATdoes not address the liver pathology so down-regulation strategiesincluding siRNA have been targeted to AAT message within hepatocytes.Since mutant AAT-PiZ exhibits a gain-of-function hepatocellular toxicityaccumulating in the endoplasmic reticulum, decreasing AAT-Pi*Z mRNAlevels (and therefore the protein) may ameliorate or even reverse theliver pathology. In addition, increased secretion of functional AATprotein will theoretically protect the lungs from neutrophil elastaseand associated proteolytic enzymes.

The strategies described herein include the development of rAAV mediatedtherapies to both augment serum levels of normal AAT and down-regulatemutant AAT using miRNA. To achieve expression and secretion of wild-typeAAT while simultaneously reducing AAT-PiZ levels. Three miRNA sequencestargeting the AAT gene were selected in some embodiments and cloned intotwo different locations of the expression cassette. The first locationis within the intron of the CB promoter driving expression of GFP, andthe second location was between the polyA sequence and the 3′ end of thegene, an additional construct with miRNAs at both locations was alsocreated. These three constructs were packaged into rAAV8 and deliveredto transgenic mice expressing the mutant form of human AAT (hAAT Pi*Z)at 6×10¹¹ vector particles per mouse via the tail vein. Theseexperiments showed about a 60% to 80% reduction in secreted AAT proteinin mice serum when compared with CB-GFP control vector injected group.It was determined that the 3×D construct was the most efficient forknocking down hAAT, in some embodiments. Liver immuno-histology alsoshowed hAAT Pi*Z protein clearance at 4 weeks after vector delivery.Using an AAT sequence with silent base pair changes to prevent the miRNAsilencing allows both up regulation of wildtype AAT gene expressionwhile simultaneously knocking down levels of mutant protein with asingle rAAV vector construct.

Materials and Methods

rAAV9 Packaging and Purification:

Recombinant AAV9 vectors used in this study were generated, purified,and titered by the UMass Gene Therapy Vector Core as previouslydescribed.

Cell Culture and Transfection:

HEK-293 cells were cultured in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum and 100 mg/l ofpenicillin-streptomycin (Gemini Bio-products Cat#400-109, Woodland,Calif.). Cells were maintained in a humidified incubator at 37° C. and5% CO2. Plasmids were transiently transfected using Lipofectamine 2000(Cat#11668-027 Invitrogen, Carlsbad, Calif.) according to themanufacturer's instructions. Cell culture supernatants or cell lyseswere collected accordingly.

Serum AAT ELISAs

Human AAT ELISA:

Total AAT protein levels were detected by ELISA. High binding extra,96-well plate (Immulon 4, cat #3855 Dynatech Laboratories, Inc.,Chantilly, Va.) were coated with 100 μl of goat anti-hAAT (1:500diluted; cat #55111MP Biomedicals, irvine CA) in Voller's bufferovernight at 4° C. After blocking with 1% non-fat dry milk in PBS-T,duplicate standard curves (hAAT; cat #16-16-011609, Athens Research andTechnology, Athens, Ga.) and serially diluted unknown samples wereincubated in the plate at room temperature for 1 hr, a second antibody,Goat anti-hAAT(HRP) (1:5000 diluted, cat #ab7635-5, Abcam Inc,Cambridge, Mass.) was incubated at room temperature for 1 h. The platewas washed with phosphate-buffered saline (PBS)-Tween 20 betweenreactions. After reaction with TMB peroxidase substrate (KPL, Inc,Gaithersburg, Md.) reactions were stopped by adding 2 N H₂SO₄ (cat#A300-500 Fisher, Pittsburgh, Pa.). Plates were read at 450 nm on aVersaMax microplate reader (Molecular Devices).

Z-AAT ELISA:

Human Z-AAT protein levels were detected by ELISA using coating antibody(1:100 diluted mouse-anti-human Alpha-1-Antitrypsin-Z, clone F50.4.1Monoclonal Antibody cat #MON5038, Cell Sciences, Inc., Canton, Mass.).Standard curves were created using PIZ mouse serum with 5% BSA (cat#B4287 Sigma, St. Louis, Mo.). Serially diluted unknown samples wereincubated in the plate at 37° C. for 1 hr, secondary antibody andfollowing the step were same as the standard human-AAT ELISA describedabove, except secondary antibody was diluted in 5% BSA and incubated inthe plate at 37° C. for 1 hr.

c-Myc ELISA:

c-Myc tag levels were quantified by a similar method as described above.Plates were coated with a c-Myc antibody (1:1000 diluted Goatanti-c-Myc, MA cat #AB 19234 Abcam, Cambridge Mass.), plates were thenblocked with 5% BSA at 37° C. for 1 hr. Standard curves were generatedfrom supernatants collected from c-Myc-AAT transfected cells.

Real-Time RT-PCR

RNA Extraction:

Flash frozen mouse liver tissue was ground up in a pestle and mortar andused to extract either small or total RNA using the mirVana miRNA RNAIsolation Kit (cat #AM 1560 Ambion, Austin, Tex.) according to themanufacturer's instructions.

microRNA qRT-PCR:

mircoRNA was primed and reverse-transcribed with TaqMan MicroRNA reversetranscription Kit (cat #4366596, Applied Biosystems Foster City,Calif.). Quantitative PCR were performed in duplicate with gene specificRT-miRNA primers and PCR Assays were designed by Applied Biosystems,using TaqMan Gene Expression Master mix (cat #436916, AppliedBiosystems, Foster City, Calif.) in a StepOne Plus real-time PCRinstrument (Applied Biosystems, Foster City, Calif.).

PIM and PIZ qRT-PCR:

Total RNA was primed with oligo(dT) and reverse-transcribed withSuperScript III First-Strand Synthesis kit for RT-PCR (Cat#18989-51,Invitrogen, Carlsbad, Calif.). Quantitative PCR were performed bygene-specific primer pairs. PIM and PIZ share the primers but differ inthe probes. Forward primer CCAAGGCCGTGCATAAGG (SEQ ID NO: 29), Reverseprimer: GGCCCCAGCAGCTTCAGT (SEQ ID NO: 30), PIZ probe:6FAM-CTGACCATCGACAAGA-MGBNFQ (SEQ ID NO: 31) and PIM probe:6FAM-CTGACCATCGACGAGA-MGBNFQ (SEQ ID NO: 32), Reactions were performedusing TaqMan Gene Expression Master mix (cat #436916, AppliedBiosystems, Foster City, Calif.) in a StepOne Plus real-time PCRinstrument (Applied Biosystems, Foster City, Calif.).

Z-AAT Transgenic Mice and rAAV9 Delivery:

The PiZ-transgenic mice used in this study have been describedpreviously. All animal procedures were performed according to theguidelines of the Institutional Animal Care and Use Committee of theUniversity of Massachusetts Medical School. Recombinant AAV9 vector wasadministered by mouse tail veil injection. The injections were performedin the most accessible vessels veins that run the length of both lateralaspects of the tail by grasping the tail at the distal end. Bleeds wereperformed through the facial vein pre-injection and every week aftertail vein rAAV9 delivery until termination of the studies.

Liver Histology:

For determination of histological changes, liver samples were fixed in10% neutral-buffered formalin (Fisher Scientific), and embedded inparaffin. Sections (5 am) were stained with hematoxylin and eosin andperiodic acid-Schiff (PAS) with or without diastase digestion.

Immuno-histochemistry for hAAT was performed as previously described¹⁴,briefly tissue sections (5 am) were deparaffinized, rehydrated, andblocked for endogenous peroxidase with 3% hydrogen peroxide in methanolfor 10 minutes. To detect hAAT expression, tissue sections wereincubated with primary antibody, rabbit antihuman AAT (1:800;RDI/Fitzgerald Industries, Concord, Mass.), for overnight at 4° C.Staining was detected using ABC-Rb-HRP and DAB kits (VectorLaboratories, Burlingame, Calif.).

Histology Image Analysis.

Slides were stained for PASD to remove glycogen. Whole digital slideimages were created using an Aperio CS ScanScope (V, CA) and analyzedusing the positive pixel count algorithm (version 9). PASD-positiveglobules were expressed as the proportion of strong positive pixels tototal pixels using a hue value of 0.9, hue width of 0.15, and colorsaturation threshold of 0.25. The intensity threshold for strongpositivity was set to an upper limit of 100.

Analysis of Z-AAT Protein Monomer and Polymer.

For soluble/insoluble protein separation, 10 mg of whole liver was addedto 2 ml buffer at 4° C. (50 mmol/l Tris-HCl (pH 8.0), 150 mmol/l NaCl, 5mmol/l KCl, 5 mmol/l MgCl2, 0.5% Triton X-100, and 80 μl of completeprotease inhibitor stock). The tissue was homogenized in a prechilledDounce homogenizer for 30 repetitions, then vortexed vigorously. A 1-mlaliquot was passed through a 28-gauge needle 10 times. The total proteinconcentration of the sample was determined, and a 5-jag total liverprotein sample was aliquoted and centrifuged at 10,000 g for 30 minutesat 4° C. Supernatant (soluble (S) fraction) was immediately removed intofresh tubes; extreme care was taken to avoid disturbing the pellet(insoluble (I) fraction). The insoluble polymers pellet (I fraction) wasdenatured and solubilized via addition of 10 l chilled cell lysis buffer(1% Triton X-100, 0.05% deoxycholate, 10 mmol/l EDTA inphosphate-buffered saline), vortexed for 30 seconds, sonicated on icefor 10 minutes and vortexed. To each soluble and insoluble sample, 2.5sample buffer (50% 5 sample buffer (5% sodium dodecyl sulfate, 50%glycerol, 0.5 mol/l Tris (pH 6.8)), 10% mercaptoethanol, 40% ddH2O) wasadded at a volume of 50% of the sample volume. Samples were boiled andloaded for sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE); equal amounts of total liver protein were loaded persoluble-insoluble pair in quantitative experiments. Densitometry wasperformed using Image J Software (NIH, Bethesda, Md.).

Serum Chemistries:

Serum samples were analyzed by UMass Mouse Phenotyping Center AnalyticalCore, using the NExCT Clinical Chemistry Analyzer (Alfa WassermannDiagnostic Technologies, West Caldwell, N.J.). Serum was analyzed foralanine aminotransferase (ALT) and aspartate aminotransferase (AST)according the manufacturers specifications.

miRNA Microarray Expression Analysis:

8 μg of total RNA were isolated from flash frozen mouse livers using themirVana miRNA isolation kit (Ambion). The experimental design includedsix groups with RNA samples from 5 mice each which were assayed onsingle color arrays for a total of 30 independent microarrays. In brief,the RNA was labeled with Cy5 and hybridized to dual-channel microarraytParaFlo microfluidics chips (LC Sciences) containing miRNA probes tomouse mature miRNAs available in the Sanger miRBase database (Release16.0) as previously described¹⁵. Each of the spotted detection probesconsisted of a nucleotide sequence complementary to a specific miRNAsequence and a long non-nucleotide spacer that extended the specificsequence away from the chip surface. Fluorescence images were collectedusing a laser scanner (GenePix 4000B, Molecular Device) and digitizedusing Array-Pro image analysis software (Media Cybernetics). The datawas analyzed including background subtraction, using a LOWESS (locallyweighted regression) method on the background-subtracted data aspreviously described¹⁶. The normalization is to remove system relatedvariations, such as sample amount variations, and signal gaindifferences of scanners. Detection was determined to be positive only iftranscripts had a signal intensity higher than 3× (background SD) andspot CV<0.5. CV as calculated by (SD)/(signal intensity), and in whichrepeating probes on the array produced signals from at least 50% of therepeating probes above detection level. Data is represented as a Log 2transformation. The data was further filtered to remove miRNAs with(normalized) intensity values below a threshold value of 32 across allsamples. t-Test were performed between “control” and “test” samplegroups where T-values are calculated for each miRNA, and p-values arecomputed from the theoretical t-distribution. If p<0.05, it is plottedas red spot in a log scatter plot.

Artificial miRNAs are as Efficient as shRNAs at Downregulating Alpha-1Antitrypsin In Vitro

Efficient Z-AAT knockdown has been demonstrated in vivo and in vitrousing shRNAs expressed from a pol III U6 promoter using rAAV8. In orderto determine if an alternative and potentially safer approach could beemployed using polymerase II driven miRNA expression, three distinctmiRNAs targeting the human AAT gene were cloned into the intron of ahybrid chicken beta-actin (CB) promoter driving GFP expression (Table 2and FIG. 1). An in vitro comparison of the previously used U6 drivenshRNAs against the pol II driven miRNAs was carried out on cell linesexpressing the human Pi*Z AAT gene. Initially a delay in Z-AAT knockdownwith the miRNAs at 24 hrs was observed, but an eventual comparable ˜35%reduction in secreted AAT protein by 48 and 72 hrs was observed for bothconstructs as compared to GFP controls (FIG. 1a ). A similar reductionwas observed in intracellular AAT protein levels assayed from the cellpellets at 72 hrs (FIG. 1b ).

rAAV9 Expressed miRNAs Mediate Efficient AAT Knockdown In Vivo

Based on the in vitro findings, the construct with the three intronicmiRNA sequences (intronic 3×miR) along with three other constructscontaining the individual miRNAs directed against the Z-AAT werepackaged in rAAV and tested in vivo in the PiZ transgenic mice. Fivegroups of 5 week old mice received: rAAV9-CB-GFP,rAAV9-CBintronic3×miR-GFP or vector with either one of the individualmiRNA via a tail vein injection with 5.0×1011 vector particles (vps) ofrAAV9. Mice were bled weekly for a total of 5 weeks to check forcirculating Z-AAT levels and were sacrificed on day 35 post rAAVdelivery. As shown in FIG. 2, mice receiving 3× intronic miRNAs(intronic 3Xmir) had on average a sustained 50-60% decrease in serum AATlevels when compared to baseline values while mice receiving the singleintronic miRNAs had on average a knockdown of 30% as compared to micereceiving the GFP control vector.

To evaluate the effect that miRNA mediated knockdown was having at theorgan level, the livers of these mice were evaluated 5 weeks post rAAVdelivery for abundance of intracellular Z-AAT. As can be appreciatedfrom liver immuno-stains for human AAT in FIG. 3, there was a markeddecrease in AAT positive staining in the livers belonging to mice in therAAV9-intronic3×miR-GFP treated group. In addition to the drasticreduction in AAT positive staining, likewise there was a dramaticdecrease in intracellular AAT globules as determined by diastaseresistant PAS (PASD) positive staining. Importantly the reduction inboth PASD and hAAT staining was accompanied by a reduction ininflammatory foci in the GFP group (FIG. 3). This suggests that thereduction in hAAT accumulation in the PiZ mice livers may be alleviatinginflammation as evidenced by the reduction in inflammatory infiltrates.

Onset and Degree of Knockdown are Dependent on miRNA Location within theExpression Cassette

While delivering 3 miRNAs within the intron of the CB promoter wassuccessful at lowering Z-AAT expression, it was unclear whether thelocation of the miRNAs within the expression cassette had any effect ontheir efficiency. It was investigated whether cloning the 3 miRNAsbetween the 3′ end of the GFP gene and the polyA tail would have aneffect on the kinetics of AAT knockdown. Likewise it was evaluatedwhether cloning the 3 miRNAs at both locations would increase (e.g.,double) the amount of miRNAs being produced and lead to a furtherenhancement of AAT knockdown. As in the previous experiments, Z-AATtransgenic mice received 5×10¹¹ vector particles of rAAV9 vectorsexpressing the miRNAs either from the intron (intronic-3×miR), polyAregion (PolyA-3×miR) or at both locations at once (Double-6×miR) (seediagram in FIG. 4). Analysis of serum Z-AAT levels revealed that by fourweeks the PolyA-3×miR and Double-6×miR were more effective than theintronic-3×miR vector at clearing serum Z-AAT levels by 85-70% and insome cases by up to 95% with the Double-6×miR vector (FIG. 4). Real-timequantitative RT-PCR analysis of liver tissue from these mice wasperformed to assay for the abundance of each of the three artificialvector derived miRs (910, 914, 943). As indicated in FIG. 5, both thePolyA-3×miR and Double-6×miR vectors produced about two-fold more copiesof each of the miRs (FIG. 5).

Having achieved a short-term clinically significant knockdown of morethan 50% of Z-AAT protein levels it was necessary to determine if thisknockdown could be sustained for longer periods of time. Once again thethree vector constructs were delivered via the tail vein at a slightlyhigher titer of 1.0×10¹² vector particles per mouse and serum Z-AATlevels were monitored weekly for 3 months. The knockdown onset of thethree vector varied within 7 weeks, the Double-6×miR vector achieved 90%knockdown 2 weeks after delivery, the PolyA-3×miR reached this mark bythe third week while the intronic-3×miR vector remained in the range of50-65% knockdown for the first 7 weeks (FIG. 6a ). Further analysis ofliver homogenates to determine whether this reduction was in the monomeror polymer pools of Z-AAT was performed on all groups. Monomer andpolymer Z-AAT fractions were separated under nondenaturing conditionsafter which, the fractions were denatured and quantitatively assessed byimmunoblotting. A reduction was observed in all groups in the monomerpool 3 months after miRNA treatment. Densitometric analysis of the bandsshowed significant differences in the PolyA-3×miR and Double-6×miR ascompared to mice treated with a control vector (FIGS. 6B-6D). Thisknockdown observed at two weeks in FIG. 6a was accompanied bysignificant reduction in serum ALT and AST in the Double-6×miR groupwith clear decreasing trends in the two other groups expressing anti-AATmiRs (FIGS. 6E and 6F). Although Z-AAT levels rose slightly for animalsin the Double-6×miR and PolyA-3×miR groups between week 7 and 13, allthree vectors stabilized at a sustained level of about 75% knockdown ofZ-AAT for the remainder of the study (FIG. 6).

In Vitro Delivery of miRNAs Against Z-AAT and Gene Correction with M-AATUsing a Single Vector

A dual-function vector that would simultaneously augment protein levelsof the wild-type M-AAT protein, thereby addressing both liver diseasecaused by the toxic gain-of-function of Z-AAT polymers and theloss-of-function caused by the absence of circulating M-AAT, wasevaluated. To achieve this, the GFP gene was replaced with a wild-typeAAT gene that had silent base pair changes at the miRNAs' target sites,thus making it impervious to the miRNA mediated knockdown. HEK-293 cellswere co-transfected with two plasmids, one of the plasmid expressedZ-AAT and the other one was either the Double-6×miR-GFP,Double-6×miR-AAT (containing the hardened, knockdown-impervious AATgene) or a control. The transfected cells were incubated for 72 hrs andRNA was harvested from cell pellets for a quantitative RT-PCR analysisof Z-AAT and M-AAT transcripts. Analysis of Z-AAT mRNA levels revealedthe both Double-6×miR-GFP and Double-6×miR-AAT produced a significantknockdown of up to 37-fold in Z-AAT mRNA copies as compared to the mocktransfected cells (FIG. 7a ). Furthermore, quantitative RT-PCR forwild-type M-AAT transcripts from the same RNA pool, revealed that theDouble-6×miR-AAT construct upregulated M-AAT expression by more than100-fold over the endogenous levels observed in control transfectedcells (FIG. 7b ).

In Vivo Delivery of Dual-Function Vectors

Taking the in vitro findings into consideration as well as the morerapid onset and the decreased variability in knockdown observed with theDouble-6×miR and PolyA-3×miR vectors (FIG. 6), both of these miRNAconfigurations were tested as dual function vectors in vivo. Threecohorts of seven mice each were dosed with 1.0×10¹² vector particleswith either a GFP control, Double-6×miR-CB-AAT or a PolyA-3×miR-CB-AATrAAV9 vectors. Serum was harvested weekly from the mice for 13 weeks andwas analyzed for Z-AAT serum levels with a PiZ specific ELISA and forM-AAT levels with an ELISA detecting the cMYC tag on the M-AAT cDNA.Changes in Z-AAT serum levels were comparable to previous experiments,with a sustained knockdown around 75-85% for both vectors (FIG. 8abottom panel). A more rapid onset of knockdown was seen with theDouble-6×miR vector but the PolyA-3×miR vector achieved similarknockdown by the fourth week. As the Z-AAT knockdown progressed, aconcomitant rise in circulating M-AAT was observed from mice receivingthe dual function vectors (FIG. 8a upper panel).

Surprisingly, while the knockdown for both vectors was similar fourweeks post delivery, the production of M-AAT was substantiallydifferent. The PolyA-3×miR-CB-AAT vector produced 8-10 times more M-AATthan the Double-6×miR-CB-AAT vector. Liver RNA was extracted from thesemice at the end of the study to quantify the mRNA levels of Z-AAT andM-AAT. A precipitous decrease in Z-AAT mRNA occurred in both cohorts ofmice receiving vectors with miRNAs as compared to mice receiving arAAV9-CB-GFP control (FIG. 8B). A quantitative RT-PCR for M-AAT was alsoperformed, to verify production of M-AAT at the RNA level and todetermine if the difference in M-AAT production between dual-functionvectors was related to mRNA transcription. Despite the clear differencein M-AAT serum protein levels, there was no statistically significantdifference in the M-AAT mRNA levels between the two groups (FIG. 8C).This indicates that mRNA processing and translation but not the level oftranscription may be affected in the Double-6×miR-CB-AAT group, in somecases.

Analysis of Global Liver miRNA Profiles after Delivery of ArtificialmiRNAs with rAAV9

A microarray analysis of endogenous mouse miRNAs from liver tissue for 6groups of mice with 5 mice per group was performed on 30 separatemicrofluidic chips using samples obtained from the long-term Z-AATknockdown experiments (FIG. 6), along with 5 untreated Z-AAT transgenicmice and 5 C57/BL6 mice. In order to determine basal differencesimparted by the human Z-AAT gene in mice, an initial comparison betweenuntreated PiZ mice and wiltype C57BL6 mice was performed. As shown inFIG. 9a and Table 3, there were only 4 statistically significantdifferences among these mice with only miR-1 having a log 2 ratiogreater than 2, being upregulated in PiZ mice. The effects ofrAAV9-CB-GFP, rAAV9-Double-6×miR-CB-GFP, rAAV9-PolyA-3×miR-CB-GFP andrAAV9-intronic-3×miR-CB-GFP liver transduction on liver miRNA profileswere compared. Surprisingly the expression of the artificial vectorderived miRNAs had minimal impact on global miRNA profiles (see FIGS.9B-9D). Statistically significant differences between untreated PiZ miceand rAAV9 treated mice were observed in 2-6 differentially expressedmiRNAs. Of these differentially expressed miRNAs the one with thelargest change was miR-1 which was down-regulated back down to levelsobserved in the C57Bl6. This correction of miR-1 up-regulation in PiZmice was observed in all groups including the mice receiving onlyrAAV9-GFP. Thus it seems to be dependent on rAAV9 delivery and not onartificial miRNA delivery.

The results presented in these examples describe a combinatorialtherapeutic approach for the treatment of both liver and lung diseasepresent in alpha-1 antitrypsin deficiency. This therapeutic approach isbased on a single dual function AAV vector to deliver both miRNAstargeting AAT for clearance of mutant mRNA along with a miRNA resistantAAT cDNA for augmentation of wild-type protein. The data presentedherein support this approach as the biological activities of the miRNAsare demonstrated both by cell culture experiments, and in vivo afternumerous experiments with tail vein delivery of rAAV9-pseudotypedvectors. Depending on the configuration of the miRNAs, a long-termknockdown of circulating serum Z-AAT in a range of 50-95% wasconsistently achieved. Furthermore, in the case of dual function vectorsthis knockdown was accompanied by equally sustained expression andsecretion of wild-type M-AAT.

Knockdown of mutant Z-AAT protein is observed in PiZ transgenic miceusing a rAAV8 vector expressing U6 driven shRNAs. Initial cell cultureexperiments determined that by 72 hours the efficiency of the miRNAsused in this study were comparable to shRNAs (FIG. 1). The in vivoexperiments described herein corroborated this finding, as a significantdecrease in Z-AAT was observed with administration of therAAV9-CBintronic3×miR-GFP rAAV9 vector (FIG. 2). These experiments alsohighlighted an enhanced effect that was obtained by using 3 anti-AATmiRNAs with different target sequences as none of the vectors with asingle miRNA achieved the level of knockdown seen when they weredelivered in combination (FIG. 2). Another biological effect aside fromZ-AAT serum reduction that was observed included a significant andwidespread decrease in the accumulation of Z-AAT within the hepatocytesand a reduction of the inflammatory lymphocyte foci within the liver(FIG. 3).

Surprisingly, anti-AAT miRNA efficacy was improved by altering thelocation of the miRNA within the expression cassette. Initial short-termexperiments demonstrated that expressing the miRNAs from the 3′ end ofthe GFP gene rather than from the intron of the CB promoter lead to a25% increase in the silencing capabilities of the miRNAs and also a tosignificant decrease in the variability of this effect. Furthermore,doubling the effective miRNA dose per vector by having the miRNAsexpressed from both locations did lead to more rapid onset of Z-AATknockdown (FIG. 4). Moreover, increased miRNA production was seen forboth the PolyA-3×miR-CB-GFP and the Double-6×miR-CB-GFP vectors ascompared to the rAAV9-intronic3×miR-GFP vector. This indicates that, insome embodiments, miRNA processing from the intron of the CB promotermay be not as efficient as from the 3′ end of the GFP gene. In otherembodiments, long-term experiments showed that initial kineticdifferences in knockdown from the three vectors wanes overtime and byeight weeks the intronic3×miR-GFP decreases in variability and augmentsin silencing efficacy.

The potency and stability of the decrease in serum Z-AAT observed invivo suggests that either of these vectors would lower Z-AAT levels inPi*ZZ patients to therapeutic levels, even below those seen in Pi*MZheterozygote patients. However, in some cases, maximal clinical benefitwould be derived from a concomitant rise in M-AAT circulation. In thisregard, the dual function vectors were designed to also deliver amiRNA-resistant M-AAT cDNA. Cell culture experiments showed thefeasibility of this strategy as was shown by a decrease in Z-AATspecific mRNA with a simultaneous rise in M-AAT using a single pro-viralplasmid (FIG. 7). These experiments supported an in vivo study of thedual function vectors. The results from those experiments confirmed thein vitro data, clearly demonstrating the feasibility of concomitantknockdown and augmentation of mutant and wild-type protein respectively.These experiments also revealed that the double configuration of miRNAshad a more rapid onset of Z-AAT knockdown but the overall efficacy overtime was comparable to the PolyA-3×miR-CB-AAT vector. In addition toimproved knockdown kinetics of the Double-6×miR-CB-AAT vector, adecreased output of M-AAT was also observed (FIG. 8a ). Initially it washypothesized that this may have been a result of decreased M-AAT mRNAproduction due to the presence of miRNA within the intron of thisconstruct, but as shown in FIG. 8c , there was a statisticallysignificant difference in M-AAT mRNA was not observed between the twogroups. While mRNA transcription and stability are not affected by thepresence of miRNAs within the intron, their translation into protein maybe hindered as observed in the decrease circulating M-AAT levels in theserum of these mice.

A consideration for a clinical therapy is an effect of artificial miRNAexpression on the endogenous miRNA profiles of the target organ. Inorder to determine if rAAV9 expressed anti-AAT miRNAs were disturbingthe endogenous miRNA profiles of the liver, the livers of 30 mice wereinterrogated at the end of the study described in FIG. 6 with a miRNAmicroarray. As can be observed from FIG. 9, neither did the delivery ofrAAV9-GFP or of the vectors expressing miRNAs have a significant impacton miRNA profiles. Notably, mir-122 which is the most abundant miRNAproduced in the liver was unaffected in any group. While some miRNAswere found to be expressed at statistically different levels among thegroups, they were mostly on the border of having a 2-fold change withone exception. Interestingly, mir-1 seemed to consistently have upwardsof a 2-fold change with rAAV9 intervention. In the case of this miRNAthe fold change with rAAV intervention was in the direction of revertingthe levels back to those found in wildtype C57BL6 mice (FIG. 9a ). Thus,in summary, miRNA profiles were unperturbed and in some cases‘corrected’ back to wildtype levels with rAAV9 delivery.

These findings indicate that other diseases states requiring thecombination of augmentation of a functional allele and suppression of amutant allele may be addressed in a similar fashion. One such example isHuntington Disease (HD), in which mutant alleles cause a severeautosomal dominant disease, but in which an allele-specific knockdownmight only be feasible if the functional allele were modified to conveyresistance to a miRNA-based knockdown. It is also significant that thesemanipulations result in minimal perturbations of endogenous miRNAprofiles. This is potentially important for considering the safety ofsingle agent miRNA-based approaches, which would be useful in otheranti-viral therapies, e.g., therapies directed against HBV or HCV. Aswith the genetic diseases considered above, these are conditions inwhich the down-regulation of target genes for prolonged periods of timemay be advantageous. Therefore, emergence of the rAAV-based miRNAplatform as a means to address these problems would be useful as well.

TABLE 2  Artificial miRNA sequences miRNA910 (SEQ ID NO: 21)5′-TAAGCTGGCAGACCTTCTGTCGTTTTGGCCACTGAGTGACGACAGA AGCTGCCAGCTTAmiRNA914 (SEQ ID NO: 22)5′-AATGTAAGCTGGCAGACCTTCGTTTTGGCCACTGACTGACGAAGGT CTCAGCTTACATTmiRNA943 (SEQ ID NO: 23)5′-ATAGGTTCCAGTAATGGACAGGTTTGGCCACTGACTGACCTGTCCA TCTGGAACCTAT

TABLE 3 Statistically significant changes in liver miRNA profiles Group1 Group 2 Mean Intensity Mean Intensity Log2 Reporter Name p-value (n =5) (n = 5) (G2/G1) B6-Control PiZ-Control mmu-miR-762 2.39E−02 525 1,0991.07 mmu-miR-23a 4.03E−02 1,247 1,591 0.35 mmu-miR-1 4.95E−02 126 2,7764.46 mmu-miR-341* 4.97E−02 4,340 2,287 −0.92 PiZ-GFP PiZ-Controlmmu-miR-1 6.03E−03 5 2.776 9.13 mmu-miR-148a 7.48E−03 1,841 1,058 −0.80mmu-miR-720 9.33E−03 1,264 3,440 1.44 mmu-miR-30c 1.03E−02 2,830 1,757−0.69 mmu-miR-146a 1.71E−02 362 175 −1.05 mmu-miR-30d 4.64E−02 627 454−0.47 PiZ-PolyA Piz-Control mmu-miR-2145 1.40E−02 573 114 −2.32mmu-miR-1 2.82E−02 22 2,776 6.95 mmu-miR-690 2.41E−02 3.071 534 −2.52mmu-miR-720 4.31E−02 1,816 3,440 0.92 PiZ-6X PiZ-Control mmu-miR-146a1.53E−02 445 175 −1.35 mmu-miR-1 3.04E−02 115 2,776 4.59

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Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated that various alterations,modifications, and improvements will readily occur to those skilled inthe art. Such alterations, modifications, and improvements are intendedto be part of this disclosure, and are intended to be within the spiritand scope of the invention. Accordingly, the foregoing description anddrawings are by way of example only and the invention is described indetail by the claims that follow.

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 1%, 5%, 10%, 15%, or 20% in either direction (greater than or lessthan) of the number unless otherwise stated or otherwise evident fromthe context (except where such number would be less than 0% or exceed100% of a possible value).

Use of ordinal terms such as “first,” “second,” “third,” etc., in theclaims to modify a claim element does not by itself connote anypriority, precedence, or order of one claim element over another or thetemporal order in which acts of a method are performed, but are usedmerely as labels to distinguish one claim element having a certain namefrom another element having a same name (but for use of the ordinalterm) to distinguish the claim elements.

The entire contents of all references, publications, abstracts, anddatabase entries cited in this specification are incorporated byreference herein.

What is claimed is:
 1. A recombinant adenoviral associated vector (rAAV vector) comprising: (a) a first region that encodes one or more first miRNAs comprising a nucleic acid having sufficient sequence complementary with an endogenous mRNA of a subject to hybridize with and inhibit expression of the endogenous mRNA, wherein the endogenous mRNA encodes a first protein having a dominant negative or gain of function mutation; and (b) a second region encoding an exogenous mRNA that encodes a second protein, wherein the second protein has an amino acid sequence that is at least 85% identical to the first protein and does not have the dominant negative or gain of function mutation, wherein the one or more first miRNAs do not comprise a nucleic acid having sufficient sequence complementary to hybridize with and inhibit expression of the exogenous mRNA, and wherein the first region is positioned between a portion of the second region encoding the last codon of the exogenous mRNA and a polyadenylation sequence of the exogenous mRNA.
 2. The rAAV vector of claim 1, further comprising a third region encoding a one or more second miRNAs comprising a nucleic acid having sufficient sequence complementary to hybridize with and inhibit expression of the endogenous mRNA, wherein the third region is positioned within an untranslated portion of the second region.
 3. The rAAV vector of claim 2, wherein the third region is between the first codon of the exogenous mRNA and a position 1000 nucleotides upstream of the first codon.
 4. The rAAV vector of claim 1, wherein the first region encodes two first miRNAs.
 5. The rAAV vector of claim 1, wherein the first region encodes three first miRNAs.
 6. The rAAV vector of claim 2, wherein the third region encodes two second miRNAs.
 7. The rAAV vector of claim 2, wherein the third region encodes three second miRNAs.
 8. The rAAV vector of claim 1, wherein the exogenous mRNA has one or more silent mutations compared with the endogenous mRNA.
 9. The rAAV vector of claim 1, further comprising an inverted terminal repeats (ITR) of an AAV serotypes selected from the group consisting of: AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, AAV11 and variants thereof.
 10. The rAAV vector of claim 1, further comprising a promoter operably linked with the region(s) encoding the one or more first miRNAs, the exogenous mRNA, and/or the one or more second miRNAs.
 11. The rAAV vector of claim 10, wherein the promoter is a tissue-specific promoter.
 12. The rAAV vector of claim 10, wherein the promoter is a β-actin promoter.
 13. A recombinant Adeno-Associated Virus (AAV) comprising an rAAV vector of claim
 1. 14. A composition comprising the recombinant AAV of claim
 13. 15. The composition of claim 14, further comprising a pharmaceutically acceptable carrier.
 16. A kit comprising a container housing the composition of claim
 14. 17. The kit of claim 16, further comprising written instructions for administering the rAAV to a subject. 